Composition and method for in vitro activation and expansion of serial killer T cell populations and passive immunization of cancer patients with tumor cell death cells

Abstract

The described invention provides a method for in vitro immunoactivation of mononuclear cells by contact with a core of three essential immunomodulatory peptides and one or more populations of genetically engineered leukocyte stimulator cells to selectively express additional R immunomodulatory peptides, and a use of a cell product comprising an expanded and activated mononuclear cell population comprising one or more subpopulations of a series of cytotoxic, apoptotic cells for passive immunization of cancer patients who are not currently under the influence of immunosuppressive therapy.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] The present application claims the benefit of priority to U.S. non-provisional application 16 / 660,442 filed on October 22, 2019, which is a partial continuation of U.S. non-provisional application 15 / 821,105 filed on November 22, 2017, which claims priority to U.S. provisional application 62 / 425,424 filed on November 22, 2016, the contents of which are incorporated by reference in their entirety.

[0003] Sequence list

[0004] The present application has been submitted electronically in ASCII format and contains a sequence list, the entirety of which is incorporated by reference into this specification. The said ASCII copy created on October 21, 2020, is named 128663-00320_SL.txt and has a size of 302,110 bytes.

[0005] Field of invention

[0006] The described invention generally relates to an immunological approach to the treatment of cancer, and more specifically to the in vitro induction and expansion of a series of killer T cell populations, followed by the passive immunization of a cancer patient with a series of expanded killer T cells in which tumor cell death is activated. Background Technology

[0007] The human immune system is a complex arrangement of cells and molecules that maintains immune homeostasis to preserve the integrity of the organism by eliminating all elements deemed dangerous. Responses in the immune system can generally be divided into two sectors called "innate immunity" and "adaptive immunity."

[0008] The innate portion of the immune system is a non-specific, rapid response to pathogens primarily responsible for the initial inflammatory response through numerous soluble factors, including the complement system and the chemokine / cytokine system; and numerous specialized cell types, including mast cells, macrophages, dendritic cells (DCs), and natural killer (NK) cells.

[0009] The adaptive immunity sector involves specific, delayed, and longer-lasting responses by various types of cells that generate long-term immunological memory for specific antigens. It can be further subdivided into cellular and humoral branches, the former primarily mediated by T cells and the latter by B cells. T cells can be further classified by the expression of CD4+ molecules or CD8+ molecules, the latter allowing for the identification of CD8+ cytotoxic T lymphocytes (CTLs).

[0010] The third sector of the immune system includes lineage members of the adaptive sector who have effector functions in the innate sector, and thus bridge the gap between the innate immune response and the adaptive immune response. These include γδ T cells and T cells with a limited T cell receptor repertoire, such as natural death T (NKT) cells and mucosa-associated constant T (MAIT) cells. The third sector will be referred to herein as "innate-like immunity."

[0011] The three sectors of immunity do not operate independently of each other but rather work together to elicit an effective immune response. Because the initiation of an adaptive immune response requires time, innate immunity and innate-like immunity provide a first line of defense during the critical period immediately after the host is exposed to a pathogen.

[0012] Components of the immune system

[0013] The immune system involves cell interactions occurring through specific receptor-ligand pairs that send signals in both directions, and each cell receives instructions based on the temporal and spatial distribution of these signals.

[0014] Cells of the immune system include lymphocytes, monocytes / macrophages, dendritic cells, closely related Langerhans cells, natural death (NK) cells, mast cells, basophils, and other members of the myeloid lineage. Additionally, a series of specialized epithelial and stromal cells provide the anatomical environment in which immunity arises by secreting important factors that regulate the growth and / or gene activation of immune system cells, and these also play a direct role in the induction and effector phases of the response. (Paul, WE, "Chapter 1: The immunological system: an introduction", Fundamental Immunology, 4th Edition, Ed. Paul, WE, Lippicott-Raven Publishers, Philadelphia, (1999), at p. 102).

[0015] Cells of the immune system are found in peripherally organized tissues such as the spleen, lymph nodes, the Fire's patches of the small intestine, and tonsils. Lymphocytes are also found in central lymphoid organs, the thymus, and the bone marrow, where they undergo developmental stages that enable them to mediate the countless responses of the mature immune system. A significant portion of lymphocytes and macrophages, including the pool of circulating cells found in the blood and lymph, provide the means to deliver immunocompetent cells to the sites where they are needed and to allow locally generated immunity to be generalized (Id.).

[0016] Leukocytes derived from the myeloid or lymphoid lineage provide innate or specific adaptive immunity. Bone marrow cells include highly phagocytic, motile neutrophils, monocytes, and macrophages, which provide a first line of defense against most pathogens. Other bone marrow cells, including eosinophils, basophils, and their corresponding tissue, mast cells, are involved in defense against parasites and the generation of allergic reactions. Lymphocytes regulate the action of other leukocytes and generate specific immune responses that prevent chronic or recurrent infections (Id.).

[0017] Complement System. Part of innate immunity, the complement system comprises more than 30 different proteins circulating in the plasma. In the absence of infection, complement proteins circulate in an inactive form. In the presence of pathogens, complement proteins become activated to kill the pathogens either directly or by promoting phagocytosis. There are two pathways through which the complement system acts on pathogens: the classical pathway, which includes antibody-dependent cell-mediated cytotoxicity; and the alternative pathway, which includes complement-dependent cell cytotoxicity. (Ricklin, Daniel, et al. "Complement: a Key System for Immune Surveillance and Homeostasis." Nature Immunology, US National Library of Medicine, Sept. 2010, www.ncbi.nlm.nih.gov / pmc / articles / PMC2924908 / ).

[0018] Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism in which effector cells of the immune system actively elute target cells bound by antibodies. The mechanism of ADCC death of antibody-coated target cells by cytotoxic effector cells occurs through non-phagic processes. This process involves the release of the contents of cytotoxic granules or the expression of cell death-inducing molecules. ADCC is triggered by the interaction between specific Fc receptor glycoproteins present on the surface of effector cells that bind to the Fc domain of immunoglobulin (Ig) and target-binding antibodies (belonging to the IgG, IgA, or IgE classes). Effector cells mediating ADCC include natural death (NK) cells, monocytes, macrophages, neutrophils, eosinophils, and dendritic cells. ADCC depends on a number of parameters, such as the density and stability of antigens on the surface of target cells, antibody affinity, and FcR-binding affinity.

[0019] Unlike ADCC, complement-dependent cytotoxicity (CDCC) is a process of the immune system that kills pathogens by damaging target cell membranes without the intervention of antibodies. This alternative pathway is initiated by the spontaneous hydrolysis and activation of complement component C3, which binds directly to the surface of microorganisms. Alternatively, the lectin pathway is initiated by soluble carbohydrate-binding proteins that bind to specific carbohydrate molecules on the surface of microorganisms.

[0020] The mechanisms of action of ADCC and CDCC, respectively, generate C3-converting enzymes that cleave C3, releasing C3a while leaving C3b bound to the surface of the pathogen. This results in numerous cellular activities, including the activation of the complement cascade, the recruitment of phagocytes to the site of infection, phagocytosis of the pathogen by immune cells, and / or the formation of Membrane Attack Complexes (MACs) that destroy the pathogen cell membrane and cause cell lysis.

[0021] immune response

[0022] Generally speaking, the immune response is initiated by an encounter between an individual and a foreign substance, such as an infectious microorganism. An infected individual responds rapidly through a humoral immune response involving the production of antibody molecules specific to the epitopes / antigens of the immunogen, and a cell-mediated immune response involving the expansion and differentiation of effector T-lymphocytes and the production of cytokines and killer T cells capable of eluting infected cells. Primary immunity using a given microorganism elicits antibodies and T cells that are specific to the epitopes / antigens found on that microorganism but typically fail to recognize or only insufficiently recognize epitopes expressed by unrelated microorganisms (Paul, WE, "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, WE, Lippicott-Raven Publishers, Philadelphia, (1999), at p. 102).

[0023] As a result of this initial response, the immunized individual develops a state of immunological memory. A secondary response follows upon a re-encounter with the same or closely related microorganism. This secondary response consists of an antibody response, which is a T-cell response that is generally faster, larger, binds to antigens with greater affinity, and is more effective in eliminating microorganisms from the body, and is similarly enhanced and often more effective. However, the immune response to an infectious agent does not always lead to the elimination of the pathogen (Paul, WE, "Chapter 1: The Immunology: Introduction," Fundamental Immunology, 4th Edition, Ed. Paul, WE, Lippicott-Raven Publishers, Philadelphia, (1999), p. 102).

[0024] Immune homeostasis

[0025] The immune system is a strictly regulated network capable of maintaining homeostasis under typical normal physiological conditions, in that its various agents work together to avoid immune imbalance. Normally, when attacked by foreign antigens, specific appropriate responses aimed at restoring balance are initiated. However, under certain circumstances, this balance is not maintained, and the immune response becomes under- or over-reacting. Cancer is an example of a situation where an inefficient or unresponsive immune response can lead to the uncontrolled growth of cancer cells. Conversely, if the immune response over-reacts, this can result in conditions such as autoimmunity, chronic inflammation, and / or post-infectious pathology.

[0026] Immune tolerance

[0027] The immune system is tolerant of self-antigens; that is, it can distinguish between antigenic determinants expressed for foreign substances and antigenic determinants expressed by host tissues. The system’s ability to ignore host antigens, known as immune tolerance or immunological tolerance, is an active process involving the elimination or inactivation of cells capable of recognizing self-antigens through immunological tolerance (Fundamental Immunology, 4th Edn, William E. Paul, Ed. Lippincott-Raven Publishers, Philadelphia, (1999), p. 2).

[0028] Innate immune cells recognize and distinguish between autogenous and non-autogenous through three distinct mechanisms: 1) Innate leukocytes can recognize "non-autogenous" from "non-infectious autogenous" by recognizing conserved products not expressed by the host; 2) Innate immune cells can recognize "lost autogenous" by recognizing autogenous proteins that are host-specific and absent in pathogens; 3) Innate immune cells can also recognize "altered autogenous" by recognizing abnormal cellular markers that are upregulated due to infection or cellular modification. (Spear, Paul, et al., "NKG2D Ligands as Therapeutic Targets.") Cancer Immunity , Academy of Cancer Immunology, 1 May 2013, www.ncbi.nlm.nih.gov / pmc / articles / PMC3700746 / ).

[0029] Immune tolerance is classified into 1) central tolerance or 2) peripheral tolerance depending on the location where the state is originally induced, namely the thymus and bone marrow (central) or other tissues and lymph nodes (peripheral). Although the biological mechanisms by which these forms of tolerance are established are distinct, the resulting effects are similar (Raker VK et al., Front Immunol, Vol., 6(569): 1-11, (2015)).

[0030] Central tolerance, the primary way the immune system is trained to distinguish between self-molecules and non-self-molecules, is established by the deletion of self-reactive lymphocyte clones before they mature into fully immunocompetent cells. This occurs during lymphocyte development in the thymus and bone marrow for T and B lymphocytes, respectively (Sprent J. et al., Philos Trans R Soc London B Biol Sci, Vol. 356(1409): 609-616, (2001)). In these tissues, mature lymphocytes are exposed to self-antigens presented by thymic epithelial cells, thymic dendritic cells, or bone marrow cells. Self-antigens are present due to endogenous expression, the influx of antigens from peripheral sites via circulating blood, and, in the case of thymic stromal cells, the expression of proteins from other non-thymic tissues through the action of the transcription factor AIRE (Murphy, Kenneth. Janeway's Immunobiology : 8th ed. Chapter 15: Garland Science. (2012), pp. 611-668; also see Klein L. Cell, Vol. 163(4):794-795, (2015). These lymphocytes, possessing receptors that bind strongly to self-antigens, are eliminated by means of apoptosis of self-reactive cells or by the induction of energy (Id. at pp. 275-334). Weakly self-reactive B cells may also remain in an immunologically inactive state, unresponsive to stimulation of their B cell receptors. Some weakly self-recognizing T cells alternatively differentiate into natural regulatory T cells (nTreg cells), which play a surveillance role in the periphery to reduce potential instances of T cell self-reactivity (Id. at pp. 611-668).

[0031] Because T cells are the major population of cells capable of causing direct tissue damage, the deletion threshold is stricter for T cells than for B cells. Furthermore, it is more advantageous for B cells to recognize a wider variety of antigens so that the organism can induce antibodies against a wider variety of pathogens. Since B cells can only be fully activated after being identified by more self-restricted T cells that recognize the same antigen, their self-reactivity is maintained at a significantly suppressed level (Id. at pp. 275-334).

[0032] This process of negative selection causes T and B cells, which could potentially initiate a potent immune response against the individual's own tissues, to be destroyed while retaining their ability to recognize foreign antigens. Lymphocyte development and education are most active during fetal development but continue throughout life as immature lymphocytes are produced, slowing down as the thymus degenerates and the bone marrow shrinks in adulthood (Id. at pp. 275-334; see also Jiang TT J Immunol., Vol. 192(11): 4949-4956, (2014)).

[0033] Peripheral tolerance occurs after T and B cells mature and enter peripheral tissues and lymph nodes (Murphy, Kenneth. Janeway's Immunobiology: 8th ed. Chapter 8: Garland Sciences. pp. 275-334). This is explained by multiple overlapping mechanisms of action, primarily involving control at the level of T cells, particularly CD4+ helper T cells, which coordinate the immune response and provide B cells with the confirmation signals necessary to proceed with antibody production. Because T cells leaving the thymus are relatively safe, though not completely safe, inappropriate reactivity to normal self-antigens that were not cleared from the thymus may occur. Some T cells will possess TCRs capable of responding to self-antigens that were not encountered in the thymus (Id.). These self-reactive T cells, which evade thymic-intrathymic negative selection in the thymus, can cause cellular damage unless they are deleted in peripheral tissues, primarily by nTreg cells.

[0034] Generally, autoimmune regulators (Aires) expressed in thymic medullary epithelial cells play a role in immune tolerance by mediating the ectopic expression of peripheral autoantigens and the deletion of self-reactive T cells (Metzger TC, et al. Immunol. Rev. 2011, 241: 89-103, (2011)).

[0035] Appropriate responsiveness to a specific antigen can also be suppressed by the induction of tolerance following repeated exposure. Naive CD4+ helper T cells differentiate into Treg cells (iTreg cells) derived from peripheral tissues, or consequently from surrounding lymphoid tissues (lymph nodes, mucosa-associated lymphoid tissues, etc.). This differentiation is mediated by IL-2 generated upon T cell activation and TGF-β from any various sources, including resistant dendritic cells (DCs) or other antigen-presenting cells (Curotto de Lafaille et al., Immunity, 30(6): 626-635, (2009)).

[0036] Immunity and Cancer

[0037] Cancer immune tolerance

[0038] While cancer is characterized by the genetic instability of specific cells, it has also been described as a disorder of the immune system based on the fact that, in at least certain segments of the affected human population, the immune system fails to respond optimally to cancerous cells that assume an clearly non-self phenotype and must be recognized as foreign. Several reasons have been proposed to explain the basis for this observation. For example, first, cancer cells are composed primarily of self-antigens, which stands in stark contrast to the situation with infectious organisms. Some antigens classified as cancer antigens are actually overexpressed normal antigens or normal antigens with mutations in only one or two amino acids in their polypeptide chains. Second, cancer cells downregulate MHC and therefore do not present many tumor cell-derived peptides via the MHC mechanism. Third, cancer cells and associated tumor-associated macrophages express cytokines that attenuate the immune response (see, e.g., Yu et al. (2007), Nature Rev. Immunol. 7:41-51). This attenuation is caused, for example, by the secretion of interleukin-10 (IL-10) by cancer cells or associated macrophages. Fourth, unlike in situations involving infection, cancer cells do not provide any adjuvants. Pathogens express various naturally occurring adjuvants that take the form of TLR agonists and NOD agonists (see, e.g., Kleinnijenhuis et al. (2011) Clin. Dev. Immunol. 405310 (page 12)). In general, optimal activation of dendritic cells requires contact between one or more TLRs expressed by dendritic cells and adjuvants. Without the activation of dendritic cells, contact between dendritic cells and T cells (immune synapses) does not result in optimal activation of T cells.

[0039] Tumor Immunological Surveillance and Immunoediting

[0040] While functional cancer immunosurveillance processes acting as exogenous tumor suppressors do exist, it has become clear that the immune system can promote tumor progression, at least partially, by sculpting the tumor's immunogenic phenotype as it develops. This so-called "tumor immune editing" is divided into three phases: the elimination phase, the equilibrium phase, and the escape phase. The elimination phase, also known as immunosurveillance, is the process in which the immune system identifies cancerous or pre-cancerous cells and eliminates them before they grow out of control. This phase can be completed when all cancerous or pre-cancerous cells are eliminated. If some tumor cells are not eliminated, a transient equilibrium may be achieved between the immune system and tumor cell growth. In this equilibrium phase, tumor cells can remain dormant or continue to evolve by accumulating additional changes to their genomic DNA to regulate existing antigens. During this process, the immune system exerts selective pressure on the evolving cells, giving tumor cells that are barely recognizable a survival advantage. Eventually, the immune response fails to recognize the tumor cells, and as a result, transitions to the escape phase, where the tumor cells grow progressively out of control. (Dunn, GP et al., Ann. Rev. Immunol. (2004): 329-60).

[0041] Tumor Immunology

[0042] Tumors can progress and evolve through numerous evasion mechanisms.

[0043] For example, tumors can evolve under selective pressure from the immune response to selectively lose receptors that activate anti-tumor immune cells. For instance, it has been reported that in NKG2D-expressing mice, NKG2D ligand-deficient tumors can persist despite the loss of other tumor cells. (Marcus, Assaf, et al. "Recognition of Tumors by the Innate Immune System and Natural Killer Cells.") Advances in Immunology , US National Library of Medicine, 2014, www.ncbi.nlm.nih.gov / pmc / articles / PMC4228931 / ).

[0044] Tumors also shed ligands that activate anti-tumor immune cells through various techniques such as alternative splicing, cleavage, proteolytic shedding, or exosome secretion. This can be observed in an increase in soluble ligands, such as MICs (MHC Class I-associated molecules that are spaced apart from MHC Class I proteins) and UL16-binding proteins (ULBPs) that bind to MICBs, and has been identified in the serum of patients with various tumor types, including breast, lung, colon, and ovarian proximal tumors, gliomas, neuroblastomas, leukemia, and melanoma. The shedding of ligands and the presence of soluble ligands in the surrounding reactive environment can result in several distinct effects. First, it reduces the susceptibility of tumor cells to attack by lymphocytes by decreasing the levels of activating ligands on the cell surface. For example, shedding of NKG2D ligands from tumor cells has been hypothesized to reduce their ability to be lysed when attacked by NK or T cells. Alternatively, the presence of soluble ligands in the reaction environment can desensitize NK cells by preventing the interactions necessary to bind to ligand receptors on lymphocytes and induce cytotoxic activity on tumor cells. Id. Soluble ligands are also thought to downregulate the expression of their receptors. For example, cancer patients with elevated serum soluble MICA levels exhibited strongly reduced NKG2D staining of their peripheral blood CD8+ T cells. Id. It has also been hypothesized that soluble ligands, along with exosomes, bind together and cooperate to influence lymphocyte immune responses. Id.

[0045] Similarly, tumors may lose the ability to express and / or shed receptors in an effort to avoid apoptosis. For example, tumors may evade immune recognition by disrupting MHC class I restricted antigen processing through the loss of class I itself or components in class I pathways. Some melanomas have lost cell surface expression of MHC class I through defective expression of β2 microglobulin (β2M), which is required for the stable assembly of class I, or through defective expression of transporters associated with tumor antigen processing (TAP). (Alberts, DS, and LM Hess, editors. FUNDAMENTALS OF CANCER PREVENTION . SPRINGER NATURE, 2019. Pps. 79-108).

[0046] Tumor microenvironment

[0047] The tumor microenvironment provides a consistently effective barrier to immune cell function because tumors actively downregulate all stages of the anti-tumor immune response using various strategies and mechanisms of action. Many molecular mechanisms causing immune cell dysfunction in the tumor microenvironment have been identified, including those directly mediated by factors generated by the tumor and others resulting from alterations in normal tissue homeostasis in the presence of cancer. Most human tumors appear capable of disrupting one or more stages of immune cell development, differentiation, migration, cytotoxicity, and other effector functions (TL Whiteside, The tumor microenvironment and its role in promoting tumor growth, Oncogene (2008) 27, 5904-5912).

[0048] One such mechanism of action involves the accumulation of regulatory T cells (Treg) (CD4+CD25bright Foxp3+) and myeloid-derived cells (CD34+CD33+CD13+CD11b+CD15-) in tumors, which are common features of human tumors, and is associated with a poor prognosis in cancer patients (Id.). Under normal conditions, Treg cells are involved in preventing autoimmunity, but in cancer, they expand and migrate into tumors, downregulate auto-effector T-cell proliferation, and suppress the anti-tumor response of both CD4+CD25- and CD8+CD25- T cells using unique molecular pathways. In tumors, Treg cells constitute a heterogeneous population of regulatory CD3+CD4+ T cells, comprising a subset of natural Tregs, antigen-specific Tr1 cells, and other poorly defined suppressor cells. T regulatory type 1 (Tr1) cells are induced in a tumor microenvironment rich in IL-10, TGF-β, and prostaglandin E2 (PGE2), all of which have been shown to promote Tr1 production (Id.).

[0049] Myeloid-derived suppressor cells (MDSCs), which are closely related to neutrophils and monocytes, do not exist in a normal state in healthy individuals but appear in cancers and pathological conditions associated with chronic inflammation or stress (Gabrilovich, DI., "Myeloid-derived suppressor cells," Cancer Immunol. Res. (2017) 5(1): 3-8). They represent a relatively stable and distinct state of functional activity of neutrophils and monocytes. The main functional characteristic of these cells is their potent ability to suppress various types of immune responses. MDSCs consist of two large cell groups: granulocytes or polymorphonuclear cells (PMN-MDSCs) that are phenotypically and morphologically similar to neutrophils; and monocytes (M-MDSCs) that are phenotypically and morphologically similar to monocytes. Therefore, phenotypic criteria alone are not sufficient to identify cells as MDSCs. In most cancer types, PMN-MDSCs account for more than 80% of all MDSCs. In addition to these two major populations, MDSCs include a small group (less than 3%) of cells with myeloid colony-forming activity representing a mixture of myeloid progenitor cells and precursors. Among peripheral blood mononuclear cells (PBMCs), PMN-MDSCs are defined as CD11b+CD14-CD15+ or CD11b+CD14-CD66b+, and M-MDSCs are defined as CD11b+CD14+HLA-DR- / loCD15-. Lin- (including CD3, CD14, CD15, CD19, and CD56) HLA-DR-CD33+ cells contain a mixed group of MDSCs comprising more immature progenitor cells. The term "early-stage MDSC" (e-MDSC) has been proposed for this latter population.

[0050] Although MDSCs are involved in the suppression of different cells of the immune system, the primary target of MDSCs is T cells. Key factors involved in MDSC-mediated immunosuppression include arginase (ARG1), iNOS, TGFβ, IL-10, COX2, the sequestration of indoleamine 2,3-deoxygenase (IDO) of cysteine, the reduction of L-selectin expression by T cells, and many others. M-MDSCs and PMN-MDSCs utilize different mechanisms of immunosuppression. M-MDSCs suppress T-cell responses in both antigen-specific and non-specific ways by utilizing mechanisms associated with the production of NO and cytokines (reviewed in *Gabrilovich, DE et al., Coordinated regulation of myeloid cells by tumors. Nat Rev Immunol. (2012) 12:253-68, Id.). On the other hand, PMN-MDSCs can suppress immune responses primarily in an antigen-specific manner. The induction of antigen-specific T-cell tolerance is one of the key characteristics of these cells (Id. citing Koehn BH, et al. GVHD-associated, inflammasome-mediated loss of function in adoptively transferred myeloid-derived suppressor cells. Blood (2015) 126:1621-8; Nagaraj S, Gupta K, Pisarev V, Kinarsky L, Sherman S, Kang L, et al. Altered recognition of antigen is a mechanism of CD8+ T cell tolerance in cancer. Nat Med. (2007) 13: 828-35). The production of reactive oxygen species (ROS) is essential for this ability.The reaction of NO and superoxide produces pernitrite peroxide (PNT), which directly inhibits T cells by nitrating T-cell receptors and reducing their responsiveness to homologous antigen-MHC complexes (citing Nagaraj S, et al., Altered recognition of antigen is a mechanism of CD8+ T cell tolerance in cancer. Nat Med. (2007) 13: 828-35, Id.). PNT also reduces the binding of antigenic peptides to MHC molecules on tumor cells (citing Lu, T. et al., Tumor-infiltrating myeloid cells induce tumor cell resistance to cytotoxic T cells in mice. J. Clinical Investigation. (2011) 121: 4015-29, Id.) and blocks T-cell migration by nitrating T-cell specific chemokines (citing Molon, B. et al., Chemokine nitration prevents intratumoral infiltration of antigen-specific T cells. J Exp Med. (2011) 208: 1949-62, Id.). In addition to immunosuppressive mechanisms, MDSCs promote tumor progression by influencing the remodeling of the tumor microenvironment and tumor angiogenesis through the production of VEGF, bFGF, Bv8, and MMP9 (Tartour, E. et al., Angiogenesis and immunity: a bidirectional link potentially relevant for the monitoring of antiangiogenic therapy and the development of novel therapeutic combination with immunotherapy. Cancer Metastasis Rev. (2011) 30: 83-95; Casella, I., et al., Autocrine-paracrine VEGF loops potentiate the maturation of megakaryocytic precursors through Flt1 receptor. Blood. (2003) 101:1316-23; Shojaei, F. et al., G-CSF-initiated myeloid cell mobilization and angiogenesis mediate tumor refractoriness to anti-VEGF therapy in mouse models. Citing Proc Natl Acad Sci US A. (2009) 106: 6742-7, Id.).

[0051] Dendritic cells found in most TMEs are immature and tunable to activate specific T cells. NKTs induce DC maturation by secreting IL-4 and IFN-γ and further upregulating CD40L. DC maturation results in increased co-stimulatory capacity through the upregulation of MHC molecules CD80 and CD86, and by the production of the pro-inflammatory cytokine IL-12 and the chemokine CCL17. The presence of chemokines attracts CCR4+ cells, including CD8+ T cells, which can then be activated by "authorized" DC cells. (Gottschalk et al. (2015) "The Role of Invariant Natural Killer T Cells in Dendritic Cell Licensing, Cross-Priming, and Memory CD8+ T Cell Generation." Front Immunol 6:379).

[0052] Typically, human tumor masses contain two types of tumor cells: one is HLA class I-positive and the other is HLA class I-negative. Effective tumor immunity requires the elimination of both types of tumor cells simultaneously. NKTs are the only cell type capable of both interacting with immature DCs to induce their maturation and enhancing the functions of both NK and CD8+ T cells. NKTs induce DC maturation, enabling DCs to present tumor antigens to CD8+ T cells. Activated CD8+ T cells can then eliminate HLA class I-positive tumor cells. NKTs also produce IFNγ, which activates NKs to kill HLA class I-negative tumor targets. (Terabe, M., & Berzofsky, JA (2012). Natural killer T cells balancing the regulation of tumor immunity. New York, NY: Springer).

[0053] NKTs can form bidirectional interactions with B cells capable of presenting lipid antigens to some NKTs via CD1d. In return, NKTs can authorize B cells to effectively prime and activate anti-tumor CTL responses and provide B-call help to enhance and maintain humoral responses. (Nair and Dhodapkar (2017). "Natural Killer T Cells in Cancer Immunotherapy." Frontiers in Immunology 8:1178).

[0054] Tumor-associated macrophages (TAMs) are prominent immunosuppressive immune cells present in the tumor microenvironment. TAMs contribute to tumor progression by promoting angiogenesis, invading tumor cells, inhibiting NK cells, and enhancing T cell responses. Some NKTs have been shown to localize with CD1d-expressing TAMs in neuroblastoma and eliminate TAMs via IL-15 and CD1d-restriction mechanisms. (Id.).

[0055] NKTs can also alter the effects of CD1d+ myeloid-derived suppressor cell (MDSC)-mediated immunosuppression. MDSCs often accumulate during tumor growth, contributing to immune escape and tumor progression. Studies have revealed that NKTs can inhibit the arginate 1 and nitrous oxide synthase-mediated inhibitory activity of MDSCs. This ability to inhibit the immunosuppressive activity of MDSCs has been reported to depend on CD1d and CD40 interactions. (Id.).

[0056] While some NKTs can promote potent anti-tumor immunity, other types are known to suppress anti-tumor immune responses and perform more regulatory roles, similar to Tregs and MDSCs. The balance between immunomodulatory and immunosuppressive NKTs can determine whether the immune response against a tumor is activated to eliminate the tumor or suppressed to allow it to grow. (Terabe, M., & Berzofsky, JA (2012). Natural killer T cells balancing the regulation of tumor immunity. New York, NY: Springer).

[0057] Some NKT types have been shown to promote the accumulation of MDSCs in tumor-carrying mice. NKTs have also been shown to suppress the anti-inflammatory functions of other NKT cell types, existing T cells, and DCs. One property of immunosuppressive NKTs is their elevated production of IL-13 and IL-4 cytokines, which can steer cytokine responses toward the tumor, primarily promoting Th2 types. Studies have revealed that immunosuppressive NKT types inhibit cytotoxic T cells through IL-13 production via the IL4R and STAT6 axes, and also induce MDSCs that produce the immunosuppressive cytokine TGF-B. (Nair and Dhodapkar (2017). "Natural Killer T Cells in Cancer Immunotherapy." Frontiers in Immunology 8:1178).

[0058] It has been hypothesized that immunosuppressive NKTs secrete IFNy instead of IL-13 when stimulated by CpG, thereby enhancing the activation and function of CD8+ cells and contributing to anti-tumor effects. (Id.) Therefore, while the balance between immunosuppressive NKTs and immunomodulatory NKTs is important for enhancing anti-tumor activity, another factor is the activating ligand of the NKT itself.

[0059] Tumor Immunotherapy

[0060] Traditional chemotherapy works by killing rapidly proliferating cells, whether normal or cancerous. Targeted therapy works by stopping or slowing the growth or spread of cancer at the cellular level by targeting specific genes, proteins, or the tissue environment of the cancer that contribute to cancer growth and survival.

[0061] For example, monoclonal antibodies block specific targets outside of cancer cells and / or in the peritumoral region. Antibody therapies such as trastuzumab (Herceptin®), which is effective against tumors overexpressing HER2 / neu protein, and cetuximab (Erbitux®), an epidermal growth factor receptor inhibitor antitumor agent, have brought about significant improvements in clinical outcomes when measured, for example, by recurrence rates, progression-free survival, and overall survival.

[0062] Small molecule drugs are designed for specific targets. For example, angiogenesis inhibitors deplete the tumor by preventing surrounding tissues from forming blood vessels (e.g., bevacizumam (Avastin®); imatinib mesylate (GLEEVEC™); tamoxifen attenuates VEGF-mediated angiogenesis (EGF-mediated anti-angiogenic effects (McNamara, DA et al., Eur. J. Surg. Oncol. (2001) 27(8): 714-718)

[0063] Immunotherapy is a type of therapy that uses substances to stimulate or suppress the immune system to help the body fight cancer, infections, and other diseases. Some types of immunotherapy target only specific cells of the immune system. Others affect the immune system more generally.

[0064] Anticancer immunotherapy has been an unachieved goal for many years. One difficulty is that target antigens are often tissue-specific molecules found in both cancer cells and normal cells, and they do not elicit immunity or exhibit non-specificity regarding apoptosis (Kaufman and Wolchok eds., General Principles of Tumor Immunotherapy, Chpt 5, 67-121 (2007)). Furthermore, tumor cells possess features that hinder immune recognition, such as the loss of expression of antigens that elicit an immune response, the absence of major histocompatibility (MHC) class II, and the downregulation of MHC class I expression. These features can lead to non-recognition of tumor cells by both CD4+ and CD8+ T cells (Id.). Tumors can also evade detection through active mechanisms of action, such as the production of immunosuppressive cytokines (Id.).

[0065] Dendritic cell vaccines are vaccines made from antigens and dendritic antigen-presenting cells (APCs). Vaccination strategies containing DCs have been developed to induce tumor-specific effector T cells capable of specifically reducing tumor masses and inducing immunological memory to control tumor recurrence. For example, DCs generated in vitro by culturing hematopoietic progenitor cells or monocytes with cytokine combinations have been tested as therapeutic vaccines in cancer patients for over 10 years (Ueno H, et al., Immunol. Rev. (2010) 234: 199-212). Treatment of metastatic prostate cancer with cipulucel-T (also known as APC 8015), a cell product based on a fusion protein of prostate acid phosphatase (PAP) and granulocyte macrophage colony-stimulating factor (GM-CSF) and simply cultured concentrated blood APCs, resulted in approximately 4-month-extended median survival in Phase III trials (Higano CS, et al., Cancer (2009) 115: 3670-3679; Kantoff PW, et al., N. Engl. J. Med. (2010) 363: 411-422). This study concluded that the DC-based vaccine is safe and can induce the expansion of circulating CD4+ T-cells and CD8+ T-cells specific to tumor antigens. As a result of this and similar studies, cipulucel-T was approved by the U.S. Food and Drug Administration (FDA) for the treatment of metastatic prostate cancer, paving the way for clinical development and regulatory pathways for next-generation cellular immunotherapy products (Palucka K and Banchereau J, Nature Reviews Cancer (April 2012) 12: 265-276).

[0066] DC-tumor cell fusions were developed to generate hybrid cells that express relevant tumor-associated antigens derived from parental tumor cells and possess the ability to process these antigens and present them to appropriate cells of the immune system. While these DC-tumor cell fusions provide a wider variety of tumor antigens, they have achieved limited success in human trials, likely due to the necessary autocomponents, product heterogeneity resulting from DC cell maturation, and changes in antigen loading (Browning, M., Antigen presenting cell / tumor cell fusion vaccines for cancer, Human Vaccines & Immunotherapeutics 9:7, 1545-1548; July 2013; Butterfield, L., Dendritic Cells in Cancer Immunotherapy Clinical Trials: Are We Making Progress?, Frontiers of Immunology, 2013 4: 454).

[0067] Immuno-checkpoint inhibitors (e.g., PD-1 and CTLA4 inhibitors) have been reported to sustain endogenous anti-cancer immune responses by blocking distinct checkpoints in the active host immune response. As used herein, the term "immuno-checkpoint" refers to an array of inhibitory pathways necessary to maintain self-tolerance, which regulate the duration and extent of the immune response to minimize damage to normal tissues. Immuno-checkpoint molecules such as PD-1, PD-L1, and CTLA-4 are cell surface signaling receptors that play a role in regulating T-cell responses in the tumor microenvironment. Tumor cells have been shown to utilize these checkpoints for the benefit of upregulating their expression and activity. Given the ability of tumor cells to direct certain immune checkpoint pathways as a mechanism of immune resistance, it has been hypothesized that checkpoint inhibitors, which bind to molecules on immune cells to activate or inactivate them, can alleviate the suppression of the immune response. Recent discoveries have identified immune checkpoints or targets such as PD-1, PD-L1, PD-L2, CTLA4, TIGIT, TIM-3, LAG-3, CCR4, OX40, OX40L, IDO, and A2AR as proteins responsible for immune evasion. Specific immune checkpoint inhibitors, including antibodies against CTLA-4, the PD-1 receptor, or its ligand PD-L1, have produced impressive results in various cancers in clinical trials, leading to FDA approvals for YERVOY™ (ipilimumab, a CTLA-4 antagonist), OPDIVO™ (nivolumab; a PD-1 antagonist), and KEYTRUDA™ (pembrolizumab; a PD-1 antagonist) in multiple tumor indications, with registration trials underway in more fields.

[0068] For example, TIGIT, a member of the Ig superfamily and an immunosuppressant receptor, is overexpressed in tumor antigen-specific CD8+ T cells and CD8+ TILs and plays a significant role in inhibiting T-cell proliferation and activation; it is involved in tumor cell immune evasion and the suppression of antiviral immune responses. The anti-TIGIT monoclonal antibody OMP-313M32 targets this immune checkpoint to prevent T-cell downregulation. Upon administration, the anti-TIGIT monoclonal antibody OMP-313M32 binds to TIGIT expressed on various immune cells, including T cells, thereby preventing the interaction of TIGIT with its ligands CD112 (Nectin-2; Poliovirus Receptor-Associated-2, PVRL2) and CD155 (Poliovirus Receptor; PVR; Nectin-like Protein 5; NECL-5). This allows CD112 and CD155 to freely interact with the co-stimulatory receptor CD226 (DNAX accessory molecule-1; DNAM-1), which is expressed on immune cells such as natural death (NK) cells and CD8-positive T cells, resulting in CD226 dimerization and CD226-mediated signaling. This activates the immune system to exert a T-cell-mediated immune response against cancer cells.

[0069] TIM-3, a transmembrane protein and immune checkpoint receptor, is associated with tumor-mediated immunosuppression. The anti-TIM-3 monoclonal antibody TSR-022, monoclonal antibodies against inhibitory T-cell receptors, T-cell immunoglobulins, and mucin domain-containing protein 3 (TIM-3; TIM3; hepatitis A virus cell receptor 2; HAVCR2), and the anti-TIM-3 antibody BMS-986258, which is an antibody against TIM-3, possess potential immune checkpoint suppression and antitumor activity. Upon administration, the anti-TIM-3 monoclonal antibody TSR-022 binds to TIM-3 expressed on specific T cells, including tumor-infiltrating lymphocytes (TILs). This eliminates T-cell suppression, activates antigen-specific T lymphocytes, and enhances cytotoxic T-cell-mediated tumor cell lysis, resulting in a reduction in tumor growth.

[0070] LAG-3 is a member of the immunoglobulin superfamily (IgSF) and binds to major histocompatibility complex (MHC) class II. LAG-3 expression in TILs is associated with tumor-mediated immunosuppression.

[0071] Relatlimab (formerly known as BMS-986016, Bristol-Myers Squibb) is a monoclonal antibody targeted at inhibitor receptor lymphocyte-activating gene-3 (LAG-3) that has potential immune checkpoint suppression and antitumor activity. Upon administration, relatlimab binds to LAG-3 on tumor-infiltrating lymphocytes (TILs), activating antigen-specific T lymphocytes and enhancing cytotoxic T-cell-mediated tumor cell lysis, which can lead to a reduction in tumor growth.

[0072] The anti-LAG-3 monoclonal antibody LAG525 is a humanized monoclonal antibody directed against the inhibitory receptor lymphocyte-activating gene-3 (LAG-3), which possesses potential immune checkpoint inhibitory and antitumor activities. Upon administration, the anti-LAG-3 monoclonal antibody LAG525 binds to LAG-3 expressed on tumor-infiltrating lymphocytes (TILs) and blocks its binding to major histocompatibility complex (MHC) class II molecules expressed on tumor cells. This activates antigen-specific T-lymphocytes and promotes cytotoxic T-cell-mediated tumor cell lysis, resulting in a reduction in tumor growth. LAG-3, a member of the immunoglobulin superfamily (IgSF) expressed on various immune cells, negatively regulates T-cell proliferation and activation. Its expression on TILs is associated with tumor-mediated immunosuppression.

[0073] The anti-LAG3 monoclonal antibody TSR-033 is a humanized, immunoglobulin G4 (IgG4) monoclonal antibody directed against inhibitory receptor lymphocyte-activating gene 3 protein (LAG3; LAG-3) with potential immune checkpoint inhibitory and antitumor activity.

[0074] MK-7684, a TIGIT-targeting agent, is a co-inhibitory molecule and an antagonist that targets the immune checkpoint inhibitor T-cell immunoglobulin (Ig) and an immune receptor tyrosine-based inhibitory motif (ITIM) domain having potential immune checkpoint inhibitory and antitumor activity (TIGIT; T-cell immunoreceptor having Ig and ITIM domains; T-cell immunoglobulin and ITIM domain). Upon administration, MK-7684 targets and binds to TIGIT expressed on various immune cells, particularly tumor-infiltrating T lymphocytes (TILs) and natural death (NK) cells, thereby preventing the interaction between TIGIT and its ligands CD112 (Nectin-2; poliovirus receptor-associated-2; PVRL2) and CD155 (poliovirus receptor; PVR; nectin-like protein 5; NECL-5), which are expressed on T cells, NK cells, and certain cancer cells. This enhances the interaction between the co-stimulatory receptor CD226 (DNAX accessory molecule-1; DNAM-1), expressed on immune cells such as NK cells and CD8+ T cells, and CD112 and CD155, and activates CD226-mediated signaling. This activates the immune system to exert a T-cell-mediated immune response against cancer cells.

[0075] However, this therapy method can be successful only when a pre-existing anti-tumor immune response is present in the patient (Pardoll, D., The blockade of immune checkpoints in cancer immunotherapy, Nature Reviews: Cancer, Vol. 12, April 2012, 253).

[0076] Chimeric Antigen Receptor T-cell therapy (CAR-T) attempts to redirect T-cells toward specific cell surface tumor antigens using synthetic biology. Genetic modification of T-cells is used to confer tumor antigen recognition through the transgenic expression of chimeric antigen receptors (CARs). CARs are engineered molecules introduced into T cells that can target them to tumor antigens (Frey, NV, Porter, DL, The Promise of Chimeric Antigen Receptor T-Cell Therapy, Oncology (2016); 30(1)) pii 219281). CAR T-cells have been shown to have some efficacy against hematological malignancies and lower-grade solid tumors. However, CAR T therapy has been shown to cause several types of toxicity, including cytokine release syndrome, neurological toxicity, non-tumor recognition, and anaphylaxis (Bonifant CL, et al., Toxicity and management in CAR T-cell therapy, Molecular Therapy ― Oncolytics (2016) 3, 16011).

[0077] Cell vaccines have also been proposed as a cancer treatment. GVAX™ is a tumor vaccine in which GM-CSF is transduced from an autologous or allogeneic population of tumor cells. It was believed that GM-CSF secretion from genetically modified tumor cells stimulates cytokine release at the vaccine site, activating antigen-presenting cells to induce a tumor-specific cellular immune response (Eager, R. & Nemunaitis, J., GM-CSF Gene-Transduced Tumor Vaccines, Molecular Therapy, Vol. 12, No. 1, 18 (July 2005)). However, GVAX™ demonstrated only a limited clinical response.

[0078] Tumor cell lines possess a broad array of antigens, many of which are common to specific tumor types, while some are shared across tumors. These tumor cell lines can be genetically engineered using many immunomodulatory components defined as a result of decades of research. An allogeneic approach to immune activation has been described in the context of these allogeneic tumor cell lines modified to express at least 2 / 3 / 4 immunomodulators.

[0079] The described invention provides an effective method for tumor cell death through the adoptive delivery of activated mononuclear cells in vitro (or in vivo). The method described herein comprises the in vitro immune activation of mononuclear cells following the co-invation of homologous engineered leukocyte stimulator cells (ENLST™ cells) encoding at least three (3) immunomodulatory peptides. Through cell contact, the mononuclear cells are stimulated to differentiate, proliferate, and acquire an activated phenotype. The activated mononuclear cells, or a subpopulation thereof consisting of a series of apoptotic cells, are useful for the passive adoptive delivery of cell products to a patient. Because the cells are activated in a physiological manner, the stimulated cells maintain the homeostatic regulatory mechanisms of that cell type. Optionally, immortalizing a subpopulation comprising a series of apoptotic cells presents the potential to generate an infinite supply.

[0080] According to one embodiment, the described invention provides a method for passive immunization following in vitro activation of a T-cell population that is cytotoxic to a cancer patient who is not currently under the influence of immunosuppressive therapy, comprising, under sterile conditions, a cell product comprising an activated and expanded population of mononuclear cells comprising a series of activated and expanded subpopulations of apoptotic cells, wherein the cell product comprises: (a) (1) isolating a population of mononuclear cells (MNCs) from a biological sample; (2) preparing a population of engineered leukocyte-stimulating cells comprising a population of tumor cells genetically engineered to express one or more tumor-specific antigens and stably express a core group of three immunomodulatory molecules, wherein the core group of the immunomodulatory molecules is an OX40 ligand (OX40L)), a CD27 ligand (CD70), and a CD28 ligand (CD28L); (3) a step of inducing an in vitro immune response by stimulating a synergistic expansion of a series of cytotoxic apoptotic cells to form an activated population of MNCs containing an activated subpopulation of a series of cytotoxic apoptotic cells in vitro; (b) a step of expanding an activated population of MNCs containing an activated subpopulation of MNCs containing an activated subpopulation of MNCs containing an activated subpopulation of MNCs containing an activated subpopulation of MNCs containing an activated subpopulation of MNCs by culturing the activated MNCs to form a cell product containing an activated and expanded population of MNCs containing an activated subpopulation of MNCs containing an activated subpopulation of MNCs in vitro; (c) a step of preparing a unit dose package containing individual doses of the activated and expanded cell product, freezing the unit dose package, andA step of storing a frozen unit dose package in a cold storage facility; (d) thawing a therapeutic amount of the frozen unit dose package containing the cell product under controlled conditions; (e) optionally combining the frozen and thawed cell product with a pharmaceutically acceptable carrier to form a pharmaceutical composition; and (f) administering a therapeutic amount of the pharmaceutical composition of (e) containing the cell product of (d) or the activated and expanded cell product to a subject, wherein the therapeutic amount is effective in reducing tumor load. According to one embodiment of the method, the amino acid sequence of the wild-type OX40 ligand codon optimized for human expression is SEQ ID NO: 108, the amino acid sequence of the wild-type CD27 ligand codon optimized for human expression is SEQ ID NO: 109, and the amino acid sequence of the wild-type CD28 ligand codon optimized for human expression is SEQ ID NO: 110, SEQ ID NO: 111, or both. According to another embodiment, a population of engineered leukocyte stimulator cells genetically engineered to express one or more tumor-specific antigens and a core group of three immunomodulatory molecules is further genetically engineered to express an additional number of immunomodulatory molecules including 3 to 25 immunomodulators ("R groups"). According to another embodiment, the CD28 ligand includes CD80, CD86, or both. According to another embodiment, engineered leukocyte stimulator cells transduced or transformed to stably express the core immunomodulator OX40 ligand, CD27 ligand, and a CD28 ligand including CD80, CD86, or both are effective in synergistically inducing 2-log expansion of activated CD8+ cells in peripheral blood mononuclear cells compared to an untransformed control cell line. According to another embodiment, a subpopulation of activated MNCs in step (b)(i) is identified and isolated by flow cytometry. According to another embodiment, the activated and expanded MNC is an NK cell population, an NKT cell population,It comprises an activated and expanded population of a series of apoptotic cells comprising one or more of a CD8 CTL cell population, a CD4 cell population, and a TCRγδ cell population. According to another embodiment, the monocyte population is derived from peripheral blood or umbilical cord blood. According to another embodiment, the monocyte population is autologous to the subject. According to another embodiment, the monocyte population is homologous to the subject. According to another embodiment, the cytotoxic apoptotic activity of the activated and expanded series of apoptotic cell populations is specific to cancer antigens on genetically engineered leukocyte-stimulating cells without affecting normal cells. According to another embodiment, the cytotoxic apoptotic activity of the activated and expanded series of apoptotic cell populations is effective in killing cancer cells regardless of cancer type without affecting normal cells. According to another embodiment, administration is performed in conjunction with a compatible inhibitor of an immune checkpoint. According to another embodiment, compatible immune checkpoints include one or more of PD-1, PD-L1, TIM-3, TIGIT, and LAG-3.

[0081] According to another aspect, the described invention comprises: (a) isolating a population of mononuclear cells (MNCs) from a biological sample; (b) preparing a population of engineered leukocyte-stimulating cells comprising a population of tumor cells genetically engineered to express one or more tumor-specific antigens and a core group of three immunomodulatory molecules, wherein the core group of the immunomodulatory peptides is an OX40 ligand (OX40L)), a CD27 ligand (CD70), and a CD28 ligand (CD28L); and (c) bringing the population of MNCs of step (a) into contact with the engineered leukocyte-stimulating cells of step (b) in vitro to form a population of activated MNCs comprising an activated subpopulation of a series of cytotoxic apoptotic cells. (d) Provides a cell product comprising an expanded and activated monocyte population comprising a series of cytotoxic activated subpopulations of apoptotic cells, produced by a process comprising the step of expanding the activated population of MNCs comprising a series of activated subpopulations of apoptotic cells in vitro by culturing the activated MNCs to form a cell product comprising an expanded and activated subpopulation of MNCs comprising a series of activated subpopulations of apoptotic cells. According to one embodiment of the cell product produced by the process, the activated and expanded MNC comprises a series of cytotoxic activated and expanded subpopulations of apoptotic cells comprising one or more of an NK cell population, an NKT cell population, a CD8 CTL cell population, a CD4 cell population, and a TCRγδ cell population. According to another embodiment of the cell product produced by the process, the series of cytotoxic apoptotic cells is tumor-destroying.According to another embodiment of the cell product produced by the process, the amino acid sequence of the wild-type OX40 ligand codon optimized for human expression is SEQ ID NO: 108, the amino acid sequence of the wild-type CD27 ligand codon optimized for human expression is SEQ ID NO: 109, and the amino acid sequence of the wild-type CD28 ligand codon optimized for human expression is SEQ ID NO: 110, SEQ ID NO: 111, or both. According to another embodiment of the cell product produced by the process, contact in step (c) is effective in synergistically inducing 2-log expansion of CD8+ cells. According to another embodiment of the cell product produced by the process, the biological sample is peripheral blood or umbilical cord blood.

[0082] These and other advantages of the invention will be apparent to those skilled in the art by referring to the following detailed description. Brief explanation of the drawing

[0083] The patent or application file includes at least one drawing executed in color. A copy of the patent or patent application publication containing the color drawing(s) will be provided by the Patent Office upon request and payment of the necessary fees. Fig. 1 It illustrates a schematic diagram of vectors 1 to 7. Fig. 2 This shows a schematic diagram of the organization of scFv-anti-biotin-G3 hinge-mIgG1 vector 1. Fig. 3 This shows a schematic diagram of the complete anti-biotin ―G3 hinge-mIgG1 vector 2. Fig. 4 This shows a schematic diagram of the tissue of sGM-CSF / ires / mFLT3L vector 3. Fig. 5 This shows a schematic diagram of the tissue of sFLT3L / ires / (FLT3 signal-GM-CSF-Tm) vector 4. Fig. 6 This shows a schematic diagram of the organization of mCD40L vector 5. Fig. 7 This shows a schematic diagram of the organization of mTNFa vector 6. Fig. 8This shows a schematic diagram of the mRANKL / ires / FLT3 signal-V5-scFV anti-biotin-Tm vector 7. Fig. 9 shows a schematic diagram of vector 44. Fig. 10 This illustrates a schematic diagram of vector 97. Fig. 11 This illustrates a schematic diagram of vector 84. Fig. 12 shows a schematic diagram of vector 29. Fig. 13 This illustrates a schematic diagram of vector 107. Fig. 14 illustrates a schematic diagram of vector 116. Fig. 15 shows a schematic diagram of vector 86. Fig. 16 This illustrates a schematic diagram of vector 18. Fig. 17 This illustrates a schematic diagram of vector 17. Fig. 18 This illustrates a schematic diagram of vector 98. Fig. 19 illustrates a schematic diagram of vector 30. Fig. 20 This illustrates a schematic diagram of vector 109. Fig. 21 This illustrates a schematic diagram of vector 106. Fig. 22 illustrates a schematic diagram of vector 16. Fig. 23 This illustrates a schematic diagram of vector 83. Fig. 24 illustrates a schematic diagram of vector 31. Fig. 25 illustrates a schematic diagram of vector 12. Fig. 26 It illustrates a schematic diagram of vector 99. Fig. 27 This illustrates a schematic diagram of vector 121. Fig. 28 This illustrates a schematic diagram of vector 105. Fig. 29 illustrates a schematic diagram of vector 32. Fig. 30 This illustrates a schematic diagram of vector 37. Fig. 31 This illustrates a schematic diagram of vector 22. Fig. 32 illustrates a schematic diagram of vector 19. Fig. 33 This illustrates a schematic diagram of vector 20. Fig. 34 shows a schematic diagram of vector 89. Fig. 35 illustrates a schematic diagram of vector 21. Fig. 36 This illustrates a schematic diagram of vector 23. Fig. 37 This illustrates a schematic diagram of vector 108. Fig. 38 This illustrates a schematic diagram of vector 15. Fig. 39 illustrates a schematic diagram of vector 124. Fig. 40 This illustrates a schematic diagram of vector 65. Fig. 41 This illustrates a schematic diagram of vector 64. Fig. 42 shows a schematic diagram of vector 88. Fig. 43 This illustrates a schematic diagram of vector 96. Fig. 44 illustrates a schematic diagram of vector 14. Fig. 45 illustrates a schematic diagram of vector 119. Fig. 46 This illustrates a schematic diagram of vector 120. Fig. 47 This illustrates a schematic diagram of vector 45. Fig. 48 This illustrates a schematic diagram of vector 60. Fig. 49 shows a schematic diagram of vector 59. Fig. 50 This illustrates a schematic diagram of vector 8. Fig. 51 This illustrates a schematic diagram of vector 128. Fig. 52 illustrates a schematic diagram of vector 35. Fig. 53a and 53b Figure 53 shows the flow cytometry forward (FSC) and side scatter (SSC) plots for size and particle size after incubating the parent strain SKMEL2 (53a) and SKMEL-2 (Fig. 53b) containing immunomodulators 14, 18, and 30 with PBMC in a mixed lymphocyte tumor response assay. In Figs. 53a and 53b, the dashed ellipses represent lymphocyte gates. Fig. 53c and Fig. 53d is a parent cell line ( Fig. 53c SKMEL2 transfected or transduced with recombinant DNA sequences encoding ) and the immunomodulators OX40 ligand (vector 14), CD27 ligand (vector 18), and CD28 ligand (vector 30 containing CD80, CD86, or both) Fig. 53dThe CD8 population after incubation of PBMCs in the mixed lymphocyte tumor response assay is plotted. In the bottom panel of the graph, dashed circles represent the CD8 gate. In PBMCs co-incubated with the parent cell line (SKMEL-2), the numbers of CD4+ and CD8+ T cells are approximately equal, whereas in PBMCs co-incubated with engineered leukocyte stimulator cells ("ENLST™ cells") engineered to express the immunomodulator OX40 ligand (Vector 14), CD27 ligand (Vector 18), and CD28 ligand (Vector 30 containing CD80, CD86, or both), there is a large approximately 2 log number of CD8+ T cells. This large increase in CD8+ T cells is evident only when all three signals are delivered simultaneously and is absent when each signal is delivered individually, thus providing an example of previously unrecognized synergistic signaling. Fig. 54a and 54b Figure 54 illustrates the results of characterizing the induction of PBMC lymphocyte populations by SK-MEL-2-derived engineered leukocyte stimulator cells (ENLST™ cells) compared to in vitro induction by unmodified SK-MEL-2 parent tumor cell lines by phase-contrast microscopy and flow cytometry. Figure 54a shows the flow cytometry of Day 9 PBMCs induced by unmodified parent 5K-MEL-2 cells (left, microscopy; right). Figure 54b shows the flow cytometry of Day 9 PBMCs induced by 5K-MEL-2-derived 14-18-30 ENLST™ cells (left, microscopy; right). In Figure 54a, the oval outline in the flow cytometry corresponds to the living unmodified SKMEL-2 parent tumor cells. In Figure 54b, the arrow indicates that the ENLST™ cells are removed by the induced PBMCs. Figs. 55a, 55b, 55c, 55d, 55e and 55fFigures 55a, 55c, and 55e illustrate the results of characterizing the tumor-destroying properties of PBMCs following in vitro activation by 14-18-30 expressing SK-MEL-2 derived ENLST™ cells into SK-MEL-2 cells, SK-MEL-28 cells, and M14 cells, as determined by flow cytometry forward (FSC) and side scatter (SSC) plots for size and particle size. Figures 55a, 55c, and 55e represent the secondary mixed lymphocyte tumor response assay on day 5, where Figure 55a shows SK-MEL-2 derived ENLST™ cells and unmodified SK-MEL-2 cells; Figure 55c shows unmodified SK-MEL-28 cells; and Figure 55e shows unmodified M14 cells. Figures 55b, 55d, and 55f represent: Figure 55b unmodified SK-MEL-2 cells; Fig. 55d, non-modified SK-MEL-28 cells; Fig. 55f shows the second mixed lymphocyte tumor response assay on day 5 with SK-MEL-2 derived ENLST™ cell-activated MNCs co-incubated with non-modified M14 cells. Fig. 56a It plots a CyTOF mass cytometry single-cell phenotypic analysis map of a PBMC population, and Figs. 56b, 56c, 56d, 56e and 56fFigure 56 illustrates visNE density contour plots of CyTOF staining following PBMC induction by immunomodulators (Figures 56c, 56d, 56e, 56f) expressing parent (Figure 56b) or SK-MEL-2-derived ENLST™ cells 9 days after the primary mixed lymphocyte tumor response assay. Figure 56b shows the PBMC subpopulation shift following induction by parent SK MEL-2 cells; note that the NK cell population and myeloid cell population are absent; Figure 56c shows the PBMC subpopulation shift following induction by ENLST™ cells transfected or transfected with Vector 3, indicating the induction of B and myeloid cells; Figure 56d shows the PBMC subpopulation shift following induction by ENLST™ cells transfected or transfected with Vectors 3 and 4, indicating the induction of B cells; FIG. 56e shows PBMC subpopulation migration following induction by ENLST™ cells transduced or transfected with vectors 3, 4 and 5, showing induction of B cells and bone marrow cells; FIG. 56f shows PBMC subpopulation migration following induction by ENLST™ cells transduced or transfected with vectors 3, 4 and 6. Figs. 57a, 57b, 57c, 57d, 57e and 57f This is demonstrated by flow cytometry (Fig. 57a) and phase contrast microscopy (Figs. 57b, 57c, 57d, 57e, and 57f) that PBMCs previously co-incubated with 14-18-30 containing ENLST™ cells can elute unaltered tumor cells. At least two distinct subpopulations of PBMCs previously activated by co-incubation with 14-18-30 ENLST™ cells are capable of lysing unaltered tumor cells. Fig. 57a Figure 1 illustrates the sorting gates for CD56, CD3, and CD8 of PBMCs following 14-18-30 and 9-day co-incubation expressing ENLST™ cells in the primary mixed lymphocyte tumor cell assay; Fig. 57b is t=0 and ( Fig. 57c ) Plot CD56+CD3+ plus undeformed SKMEL2 at time t=8; Fig. 57d is t=0 and ( Fig. 57e ) Plot CD56-CD3+CD8+ plus undeformed SK-MEL-2 at time t=8. Fig. 57b and Fig. 57d The yellow arrows indicate that the smaller cells are lymphocytes and the larger cells are allogeneic tumor cells. Fig. 57c and Fig. 57e The yellow arrow indicates a cluster of lysed cells surrounding the allogeneic tumor cells, and the background indicates that the allogeneic tumor cells have been removed. Fig. 58 Figure 1 is a box and whisker plot showing the results of a xenograft therapy study using NGS mice. The ends of each box represent the upper and lower quartiles; the median is indicated by a vertical line inside the box, and the whiskers are two lines outside the box extending to the highest and lowest observations. Human tumor cells were transplanted into the flanks of NGS (NOD scid gamma) mice. The tumors were allowed to grow to 150 mm³. The mice were divided into two groups of six mice each: a control group and a treatment group. On day 30 (t=0), the control group mice were inoculated with vehicle only, while the treatment group mice were inoculated with 3 x 10⁶ cells activated by 14-18-30 expressing ENLST™ cells ("SUPLEXA™ cells"). 6 PBMC was administered. Tumor size was measured at intervals of 36 days after inoculation. Differences between the two groups appeared within 5 days. After day 22, divergence became statistically significant ( * P<0.05; ** P<005). Specific details for implementing the invention

[0084] definition

[0085] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, a reference to "peptide" refers to one or more peptides and their equivalents, etc., known to those skilled in the art.

[0086] As used herein, the term “about” means plus or minus 10% of the numerical value of the number in which it is used. Thus, about 50% means a range of 40%-60%.

[0087] The term "activation" or "lymphocyte activation" refers to the stimulation of lymphocytes by specific antigens, non-specific mitogens, or allogeneic cells, resulting in the synthesis of RNA, proteins, and DNA and the production of lymphokines; which are then followed by the proliferation and differentiation of various effector and memory cells. For example, mature B cells can be activated by encountering an antigen that expresses an epitope recognized by their cell surface immunoglobulin Ig. The activation process can be a direct process relying on the cross-linking of membrane Ig molecules by the antigen (cross-linking-dependent B cell activation) or an indirect process occurring most efficiently in the context of intimate interactions with helper T cells ("alien helper process"). T-cell activation relies on the interaction of the TCR / CD3 complex with its allogeneic ligand, which is a peptide bound to the groove of a class I or class II MHC molecule. The molecular events established by receptor binding are complex. During the early stages, there appears to be activation of tyrosine kinases that induce tyrosine phosphorylation of a set of substrates controlling various signaling pathways. This includes a set of adapter proteins that link TCRs to the ras pathway, phospholipase Cγ1, tyrosine phosphorylation that increases its catalytic activity and participates in the inositol phospholipid metabolic pathway, leading to an increase in intracellular free calcium concentration and activation of protein kinase C, and a series of other enzymes that regulate cell growth and differentiation. Full responsiveness of T cells requires, in addition to receptor junctions, assisted cell-transmitted co-stimulatory activity, e.g., junctions of CD28 on T cells with CD80 and / or CD86 on APCs. The soluble products of activated B lymphocytes are immunoglobulins (antibodies). The soluble products of activated T lymphocytes are lymphokines.

[0088] As used herein, the term "active immunization" refers to the generation of active immunity, which means immunity resulting from naturally acquired infection or intentional vaccination (artificial active immunity). Active immunity may be induced by natural or artificial mechanisms of action.

[0089] When applied to mammals, cells, tissues, organs, or biological fluids, the term “administration” and its various grammatical forms as used herein refer, without limitation, to contact of an exogenous ligand, reagent, placebo, small molecule, pharmaceutical, therapeutic agent, diagnostic agent, or composition with the subject, cell, tissue, organ, or biological fluid, etc. “Administration” may refer, for example, to treatment, pharmacokinetics, diagnosis, research, placebo, and experimental methods. “Administration” also encompasses the treatment of cells, for example, in vitro and in vitro, by reagents, diagnostics, conjugated compositions, or by other cells.

[0090] As used herein, the term “allogeneic” means that the donor and the recipient are of the same species, although their genetic makeup differs. As used herein, “allogeneic cell” refers to a cell that is not derived from the individual to which the cell is to be administered, that is, a cell having a genetic makeup different from that of the recipient individual. Allogeneic cells are generally obtained from the same species as the recipient individual to which the cell is administered. For example, allogeneic cells may be human cells as disclosed herein for administration to human patients. As used herein, the term “allogeneic series of apoptotic cell population” refers to a series of apoptotic cell populations comprising constituent cell types (e.g., NK, NKT, and CTL) derived from a donor having a genetic makeup different from that of the recipient individual to which the allogeneic series of apoptotic cell population is administered.

[0091] As used herein, the term “allogeneic recognition” refers to the recognition by T cells of MHC molecules other than self (HLA in humans). As used herein, the term “direct allogeneic recognition” refers to the process in which CD4+ and CD8+ T cells recognize either one of the intact allogeneic HLA molecules (HLA classes II and I, respectively) on a donor antigen-presenting cell (APC). The term “indirect allogeneic recognition” refers to the process in which an APC first engulfs a donor cell and then processes the donor antigen to re-mark it to the recipient immune system; and then the recipient T cell responds to the donor HLA peptide processed in the context of the recipient HLA molecule.

[0092] The terms “amino acid residue,” “amino acid,” or “residue” are used interchangeably to refer to amino acids incorporated into proteins, polypeptides, or peptides, including but not limited to naturally occurring amino acids and known analogs of naturally occurring amino acids capable of functioning in a manner similar to naturally occurring amino acids. Amino acids may be L- or D-amino acids. Amino acids may be replaced by synthetic amino acids that are modified to increase the half-life of the peptide, increase the potency of the peptide, or increase the bioavailability of the peptide. Single-character symbols for amino acids are primarily used herein. These single-character symbols are as follows: A is alanine; C is cysteine; D is aspartic acid; E is glutamic acid; F is phenylalanine; G is glycine; H is histidine; I is isoleucine; K is lysine; L is leucine; M is methionine; N is asparagine; P is proline; Q is glutamine; R is arginine; S is serine; T is threonine; V is valine; W is tryptophan; and Y is tyrosine. The following presents a group of amino acids that are conservatively substituting for one another: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

[0093] As used herein, the terms “antigen-presenting cell” or “APC” refer to highly differentiated cells capable of processing antigens and displaying peptide fragments thereof on their cell surface along with other co-stimulating proteins necessary to activate naive T cells. The major antigen-presenting cells are dendritic cells (DCs), macrophages, and B cells.

[0094] As used in this specification, the term "self" means derived from the same entity.

[0095] As used herein, the term “autocrine signaling” refers to a type of cell signaling in which a cell secretes signaling molecules that act on itself or on other adjacent cells of the same type.

[0096] The term "bond" and its other grammatical forms refer to a sustained attraction between chemical substances.

[0097] The term "binding specificity" encompasses both binding to a specific partner and binding to other molecules. Functionally important bindings can occur across a range of low to high affinities, and design elements can suppress unwanted cross-interactions. Post-translational modifications can also alter the chemistry and structure of interactions. "Crowded binding" may involve a degree of structural plasticity, which can result in different subsets of residues important for binding to other partners. "Relative binding specificity" is a characteristic in biochemical systems where a molecule interacts differentially with its target or partner, having distinct effects depending on the identity of the individual target or partner.

[0098] CD3 (TCR complex) is a protein complex composed of four distinct chains. In mammals, the complex contains the CD3γ chain, CD3δ chain, and two CD3ε chains, which link with the T cell receptor (TCR) and ζ-chain to generate activation signals in T lymphocytes. The TCR, ζ-chain, and CD3 molecules together constitute the TCR complex. The intracellular tail of the CD3 molecule contains a conserved motif known as the immune receptor tyrosine-based activation motif (ITAM), which is essential for the signaling ability of the TCR. Through the phosphorylation of ITAM, the CD3 chain can bind to ZAP70 (zeta-associated protein), a kinase involved in the T cell signaling cascade.

[0099] As used herein, the term "cell line" means a permanently established cell culture consisting of a population of cells having a uniform genetic and functional composition that develops from a single cell and thus proliferates indefinitely.

[0100] As used herein, the term “chemokine” refers to chemotactic cytokines comprising a family of structurally related proteins of low molecular weight (8-11 kDa) having various immune and neurological functions (Mackay CR Nat Immunol., Vol. 2: 95-101, (2001); Yun B. et al. Immunol Rev. (2000) Vol. 177: 150-174), which can be classified into four subfamilies (C, CC, CXC, and CX3C) based on the relative positions of conserved cysteine ​​residues (Rossi D. et al. Annu Rev Immunol. (2000) 18: 217-242). Chemokines are essential molecules for directing the movement of leukocytes between blood, lymph nodes, and tissues. Because it is not always limited to a single type of receptor, it forms a complex signaling network (Loetscher P. et al. J. Biol. Chem. (2001). 276: 2986-2991). Chemokines affect cells by activating surface receptors, which are seven-transmembrane-domain G-protein-coupled receptors. The leukocyte response to a specific chemokine is determined by the expression of that chemokine receptor. The binding of chemokines to receptors activates various signaling cascades similar to the action of cytokines, culminating in the activation of a biological response. The secretion of ligands to the CCR5 receptor, the expression and secretion of normal T cells (RANTES) and macrophage inflammatory proteins (MIP)-1α / and MIP-1β regulated upon activation (Schrum S. et al., J Immunol. (1996) 157: 3598-3604), and the ligand to the CXC chemokine receptor 3 (CXCR3), the induced protein (IP)-10 (Taub DD et al., J Exp Med. (1993) 177:1809-1814) were associated with unwanted enhanced TH1 responses. Additionally, elevated levels of the injury-prone inflammatory cytokines IL-2 and IFN-γ are correlated with type 1 diabetes (T1D) (Rabinovitch A.et al. Cell Biochem Biophys. (2007) 48(2-3): 159-63). Chemokines have been observed in TH1 pancreatic infiltrates and other inflammatory lesions characterized by T cell infiltration (Bradley LM et al. J Immunol. (1999). 162:2511-2520).

[0101] As used in this specification, the term "chemonive" means having or indicating no experience with chemotherapy.

[0102] As used in this specification, the term "chemotherapy" refers to a treatment that uses drugs to stop the growth of cancer cells.

[0103] As used in this specification, the term "composition" refers to an aggregate material formed of two or more materials.

[0104] As used herein, the term “contact” and its various grammatical forms refer to a state or condition of touching or being immediately or locally close. Contact of the composition with a target destination may be achieved by any means of administration known to those skilled in the art.

[0105] As used herein, the term “co-stimulatory molecule” refers to a molecule displayed on the cell surface that serves to enhance the activation of T cells already being stimulated via their TCRs. For example, an HLA protein presenting an alien antigen to a T cell receptor requires a co-stimulatory protein that binds to a complementary receptor on the surface of the T cell to enhance T cell activation. As used herein, the term “co-stimulatory molecule” refers to a highly active immunomodulatory protein that plays a crucial role in the development and maintenance of adaptive immune responses (Kaufman and Wolchok eds., General Principles of Tumor Immunotherapy, Chpt 5, 67-121 (2007)). The two-signal hypothesis of T cell responses includes the interaction between an antigen bound to an HLA molecule and its cognate T cell receptor (TCR), and the interaction between a co-stimulatory molecule and its ligand. Differentiated APCs, which are carriers of the second co-stimulatory signal, can activate a T cell response following the binding of the HLA molecule to the TCR. In contrast, somatic cells do not express a second signal, thereby inducing T cell non-response (Id.). Many co-stimulatory molecules involved in the two-signaling model can be blocked by co-inhibitory molecules expressed by normal tissues (Id.). In fact, many types of interacting immunomodulatory molecules expressed in a wide variety of tissues can exert both stimulatory and inhibitory functions depending on the immunological context (Id.).

[0106] As used herein, the term “cytokine” refers to a small, soluble protein substance secreted by a cell that exerts various effects on other cells. Cytokines mediate many important physiological functions, including growth, development, wound healing, and immune responses. They act by binding to their cell-specific receptors located on the cell membrane, initiating distinct signal transduction cascades in the cell, which will eventually lead to biochemical and phenotypic changes in the target cell. Cytokines can act both locally and remotely from their release sites. They include type I cytokines, which encompass many interleukins; several hematopoietic growth factors; type II cytokines, which include interferon and interleukin-10; tumor necrosis factor (“TNF”)-related molecules, including TNFα and lymphotoxins; members of the immunoglobulin superfamily, including interleukin 1 (“IL-1”); and chemokines, a family of molecules that play important roles in various immune and inflammatory functions. The same cytokine may have different effects on a cell depending on the cell's state. Cytokines often regulate the expression of other cytokines and trigger cascades. Non-limiting examples of cytokines include, for instance, IL-1α, IL-β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12 / IL-23 P40, IL13, IL-15, IL-17, IL-18, IL-21, IL-23, TGF-β, IFN-γ, GM-CSF, Gro-α, MCP-1, and TNF-α.

[0107] As used herein, the term “derived from” encompasses any method of receiving, obtaining, or modifying something from a source.

[0108] The terms “derivative” or “variant” in relation to peptide or DNA sequences (e.g., immunomodulator peptide sequences) as used herein refer to a non-identical peptide or DNA sequence modified from its original sequence. In relation to cells as used herein, the terms “derivative” or “variant” refer to a tumor cell line modified from its origin cell line (e.g., modified to express a recombinant DNA sequence).

[0109] The term "detectable marker" encompasses both selectable markers and assay markers. The term "selectable marker" refers to various gene products that allow cells transformed into expression structures to be selected or screened, including drug-resistant markers, antigen markers useful for fluorescence-activated cell classification, and adhesion markers such as receptors for adhesion ligands that allow selective attachment.

[0110] The term “detectable reaction” refers to any signal or reaction that can be detected in a assay performed with or without the use of a detection reagent. Detectable reactions include, but are not limited to, radioactive decay and the emission, absorption, polarization, fluorescence, phosphorescence, transmission, reflection, or resonance transfer of energy (e.g., fluorescence, ultraviolet, infrared, visible light). Detectable reactions also include chromatographic mobility, turbidity, electrophoretic mobility, mass spectra, ultraviolet spectra, infrared spectra, nuclear magnetic resonance spectra, and X-ray diffraction. Alternatively, a detectable reaction may be the result of an assay to measure one or more properties of a biological material, such as melting point, density, conductivity, surface acoustic waves, catalytic activity, or elemental composition. A “detectable reagent” is any molecule that produces a detectable reaction indicating the presence or absence of a substance of interest. Detection reagents include various molecules such as antibodies, nucleic acid sequences, and enzymes. To facilitate detection, detection reagents may include markers.

[0111] As used herein, the term "dosage" refers to the amount of therapeutic substance prescribed to be taken at one time.

[0112] As used herein, the term "dyes" (also referred to as "fluorescent dyes" or "fluorescent groups") refers to a component of a molecule that causes the molecule to be fluorescent. A component is a functional group in a molecule that absorbs energy at a specific wavelength and re-emits energy at a different (but equally specific) wavelength. The amount of emitted energy and the wavelength depend on both the dye and its chemical environment. FITC, R-Phycoerythrin (PE), PE-Texas Red Tandem, PE-Cy5 Tandem, Propidium Iodidem, EGFP, EYGP, ECF, DsRed, Allophycocyanin (APC), PerCp, SYTOX Green, Coumarin, Alexa Fluorus (350, 430, 488, 532, 546, 555, 568, 594, 633, 647, 660, 680, 700, 750), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Hoechst 33342, DAPI, Hoechst 33258, SYTOX Blue, Chromomycin A3, Mitramycin, YOYO-1, SYTOX Orange, Ethidium Bromide, 7-AAD, Acridine Orange, TOTO-1, TO-PRO-1, Thiazole Orange, TOTO-3, TO-PRO-3, Thiazole Orange, Propidium Iodide (PI), LDS 751, Indo-1, Fluo-3, DCFH, DHR, SNARF, Y66F, Y66H, EBFP, GFPuv, ECFP, GFP, AmCyanl, Y77W, S65A, S65C, S65L, S65T, ZsGreenl, ZsYellowl, DsRed2, DsRed Monomer, AsRed2, mRFP1, HcRedl, Many dyes are known, including but not limited to monochlorobiman, calcein, DyLight Fluors, cyanine, hydroxycoumarin, aminocoumarin, methoxycoumarin, Cascade Blue, Lucifer Yellow, NBD, PE-Cy5 conjugate, PE-Cy7 conjugate, APC-Cy7 conjugate, Red 613, fluorescein, FluorX, BODIDY-FL, TRITC, X-rhodamine, risamine rhodamine B, Texas Red, TruRed, and derivatives thereof.

[0113] As used herein, the term “engineered leukocyte stimulator cell” (or “ENLST™ cell”) refers to a homologous primary tumor cell line transfected or transduced with a recombinant DNA sequence encoding at least three core immunomodulatory peptides—the OX40 ligand, CD27 ligand, and CD28 ligand, comprising CD80, CD86, or both.

[0114] As used herein, the term “enrich” refers to increasing the proportion of a desired substance, for example, increasing the relative frequency of a cell subtype compared to its natural frequency in a cell population. Positive selection, negative selection, or both are generally considered necessary for any enrichment scheme. Selection methods include, without limitation, self-separation and fluorescence-activated cell sorting (FACS). Regardless of the specific technique used for enrichment, the specific markers used in the selection process are important because developmental stages and activation-specific responses can alter the antigen profile of cells.

[0115] As used herein, the term "expression" encompasses the biosynthesis of mRNA, polypeptide biosynthesis, polypeptide activation by, for example, post-translational modification, or activation of expression by altering subcellular location or recruitment into chromatin.

[0116] The term "expression vector" refers to a DNA molecule containing a gene that is expressed in a host cell. Typically, gene expression is placed under the control of specific regulatory elements, including but not limited to promoters, tissue-specific regulatory elements, and enhancers. These genes are referred to as being "operably linked" to the regulatory elements.

[0117] As used herein, the term "flow cytometry" refers to a tool for investigating the phenotype and characteristics of cells. It detects the movement of cells or particles in a liquid flow through a laser (light amplification by stimulated emission of radiation) / light beam passing through a sensing area. The relative light-scattering and color-identifying fluorescence of microparticles are measured. Flow analysis and differentiation of cells are based on size, particle size, and whether the cells carry fluorescent molecules in the form of antibodies or dyes. As cells pass through the laser beam, light is scattered in all directions, and light scattered forward at a low angle (0.5–10°) from the axis is proportional to the square of the radius of the sphere and thus proportional to the size of the cell or particle. Light can enter the cell; therefore, 90° light scattering (right angle, lateral) can be labeled with fluorescent dye-linked antibodies or stained with fluorescent membranes, cytoplasm, or nuclear dyes. Thus, differentiation of cell types, the presence of membrane receptors and antigens, membrane potential, pH, enzyme activity, and DNA content can be easily determined. Flow cytometry records multiple measurements for each cell using multiple parameters; thus, it is possible to identify homologous subpopulations within heterologous populations (Marion G. Macey, Flow cytometry: principles and applications, Humana Press, 2007). Fluorescence-activated cell sorting (FACS), which can isolate distinct cell populations that cannot be distinguished by size or density because their physical characteristics are too similar, uses fluorescent tags to detect differentially expressed surface proteins, thereby enabling fine distinctions among physically homologous cell populations.

[0118] The terms “functional equivalent” or “functionally equivalent” are used interchangeably herein to refer to a substance, molecule, polynucleotide, protein, peptide, or polypeptide having a similar or identical effect or use.

[0119] The term “irregularly modified” is used herein to refer to a variant peptide having higher biological efficacy than the original peptide. An “irregularly modified immunogen” is an immunogen that elicits an immune response that cross-reacts with an original non-immunogenic or poorly immunogenic antigen.

[0120] The terms “immune response” and “immune-mediated” are used interchangeably herein to refer to any functional expression of a subject’s immune system toward foreign or auto-antigens, regardless of whether the result of such response is beneficial or harmful to the subject.

[0121] As used in this specification, the term "immunogen" and its various grammatical forms refer to a substance that induces an immune response.

[0122] The terms “immunomodulation,” “immunomodulator,” and “immunomodulation” are used interchangeably herein to refer to substances, agents, or cells capable of directly or indirectly enhancing or reducing an immune response by, for example, expressing chemokines, cytokines, and other mediators of the immune response.

[0123] The term “immunostimulating amount” as used herein refers to an amount of an immunogenic composition effective in stimulating an immune response in a measurable amount, as measured by, for example, the ELISPOT assay (cellular immune response), ICS (intracellular cytokine staining assay), and major histocompatibility complex (MHC) tetramer assay for detecting and quantifying antigen-specific T cells, quantifying a blood population of antigen-specific CD4+ T cells, or quantifying a blood population of antigen-specific CD8+ T cells, or, where the increase is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% compared to an appropriate control.

[0124] In relation to immunity, the term "induce" and its various grammatical forms as used in this specification refer to a process or action that causes or results in an immune response.

[0125] As used in this specification, the term "inhibitor" refers to a second molecule that binds to, contacts, or otherwise interferes with the activity of the first molecule, thereby reducing the activity of the first molecule.

[0126] As used herein, the term "incorporate into the genome" refers to genomic DNA comprising the genome of a host cell and a recombinant DNA sequence incidentally linked thereto.

[0127] The term “isolated” is used herein to refer to (1) a substance, such as nucleic acids, peptides, or cells, but not limited to, which is substantially or essentially devoid of the components that would typically accompany or interact with it when found in its naturally occurring environment. The terms “substantially devoid” or “essentially devoid” are used herein to refer to a substance that is substantially or significantly devoid of other substances, or that is devoid of more than about 95%, 96%, 97%, 98%, 99%, or 100%. An isolated substance may optionally contain substances not found together with the substance in its natural environment; or (2) where the substance is in its natural environment, the substance is synthetically (non-naturally) altered in composition by intentional human intervention and / or placed in an intracellular location (e.g., genome or organelle of a cellular genomic cell) that is not inherent to the substance found in that environment. Alterations to create a synthetic substance may be performed on the substance in its natural state or removed from it.

[0128] As used herein, the term “labeling” refers to the process of distinguishing compounds, structures, proteins, peptides, antibodies, cells, or cellular components by introducing traceable components. Common traceable components include, but are not limited to, fluorescent antibodies, fluorescent dyes, dyes or fluorescent dyes, stains or fluorescent stains, markers, fluorescent markers, chemical stains, differential stains, differential labels, and radioisotopes.

[0129] The term "lymphocyte" refers to small white blood cells (leukocytes) formed in lymphoid tissues throughout the body and in normal adults, constituting approximately 22–28% of the total number of white blood cells in circulating blood, and playing a significant role in defending the body against disease. Individual lymphocytes are specialized in that they are committed to responding to a limited set of structurally related antigens through the recombination of their genetic material. This commitment, which exists prior to the immune system's initial contact with a given antigen, is expressed by the presence of receptors specific to determinants (epitopes) on the antigen's surface membrane. Each lymphocyte possesses a unique population of receptors, all of which share the same combination site. Since one set or clone of lymphocytes differs from another clone in the structure of its receptor combination site, the epitopes it can recognize differ. Lymphocytes differ from one another not only in the specificity of their receptors but also in their functions (Id.).

[0130] Two broad classes of lymphocytes are recognized: B-lymphocytes (B-cells) and T-lymphocytes (T-cells), which are precursors of antibody-secreting cells.

[0131] B-lymphocytes

[0132] B lymphocytes originate from hematopoietic cells in the bone marrow. Mature B cells can be activated by antigens expressing epitopes recognized by their cell surfaces. The activation process can be direct (cross-linking-dependent B-cell activation) by relying on the cross-linking of membrane immunoglobulin (Ig) molecules by the antigen, or indirect through interaction with helper T cells in a process referred to as homologous assistance. In many physiological situations, receptor cross-linking stimulation and homologous assistance produce a synergistic effect, yielding a more vigorous B-cell response (Paul, WE, "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, WE, Lippicott-Raven Publishers, Philadelphia, (1999)).

[0133] Cross-linkage-dependent B-cell activation requires that antigens express multiple copies of epitopes complementary to the binding sites of cell surface receptors, as each B-cell expresses an Ig molecule with the same variable region. This requirement is met by other antigens having repetitive epitopes, such as the capsule polysaccharides of microbial or viral envelope proteins. Cross-linkage-dependent B-cell activation is the primary protective immune response to these microorganisms (Id.).

[0134] Homomorphic help enables B cells to elicit a response to antigens that cannot cross-link to their receptors, while simultaneously providing a co-stimulatory signal that rescues B cells from inactivation when stimulated by weak cross-linking events. Homomorphic help relies on the binding of antigens by B cell membrane Ig, the endocytosis of the antigens, and their fragmentation into peptides within the cell's endosome / lysosome compartments. Some of the generated peptides are loaded into grooves of a differentiated set of cell surface proteins known as Class II major histocompatibility complex (MHC) molecules. The generated Class II / peptide complexes are expressed on the cell surface and CD4 + It acts as a ligand for antigen-specific receptors of a set of T-cells designated as T cells. CD4 + T cells carry receptors specific to the class II / peptide complex of B cells on their surface. B cell activation depends not only on the binding of the T cell to its T cell receptor (TCR), but this interaction also allows an activating ligand (CD40 ligand) on the T cell to bind to the receptor (CD40) on the B cell for signaling B cell activation. Additionally, T helper cells secrete various cytokines that regulate the growth and differentiation of B cells stimulated by binding to cytokine receptors on B cells (Id.).

[0135] During homologous assistance for antibody production, the CD40 ligand (CD40L) activates CD4 +It is transiently expressed in T helper cells and binds to CD40 on antigen-specific B cells to transduce a second co-stimulatory signal. The latter signal is essential for B cell growth and differentiation, as well as for the generation of memory B cells by preventing apoptosis of germinal B cells that encounter antigens. Overexpression of CD40L in both B and T cells is associated with the production of pathogenic autoantibodies in human SLE patients (Desai-Mehta, A. et al. J. Clin. Invest. Vol. 97(9), 2063-2073, (1996)).

[0136] T-lymphocytes

[0137] T-lymphocytes derived from precursors in hematopoietic tissues undergo differentiation in the thymus and are then seeded into peripheral lymphoid tissues and the lymphocyte recirculation pool. T-lymphocytes, or T cells, mediate a wide range of immune functions. These include the ability to assist B cells in developing into antibody-producing cells, the ability to enhance the bactericidal activity of monocytes and macrophages, the suppression of specific types of immune responses, the direct death of target cells, and the mobilization of inflammatory responses. These effects depend on the T-cell expression of specific cell surface molecules and the secretion of cytokines (Paul, WE, "Chapter 1: The immune system: an introduction", Fundamental Immunology, 4th Edition, Ed. Paul, WE, Lippicott-Raven Publishers, Philadelphia, (1999)).

[0138] T cells differ from B cells in the mechanism of their antigen recognition. Immunoglobulins, the receptors of B cells, bind to individual epitopes on the surface of soluble molecules or microparticles. Therefore, B-cell receptors detect epitopes expressed on the surface of natural molecules. While antibodies and B-cell receptors have evolved to bind to and protect microorganisms in the extracellular fluid, T cells recognize antigens on the surface of other cells and mediate their function by interacting with these antigen-presenting cells (APCs) and altering their behavior. There are three main types of APCs in peripheral lymphoid organs capable of activating T cells: dendritic cells (DCs), macrophages, and B cells. The most potent of these is the DC, whose sole function is to present foreign antigens to T cells. Immature DCs are located in tissues throughout the body, including the skin, intestines, and airways. When they encounter invading microorganisms in these areas, they endocytose pathogens and their products, transporting them via the lymph to local lymph nodes or intestinal lymphoid organs. Encounter with pathogens induces the DC to mature from an antigen-capturing cell into an APC capable of activating T cells. APCs display three types of protein molecules on their surface that serve to activate T cells and turn them into T effector cells: (1) HLA proteins that present foreign antigens to T cell receptors; (2) co-stimulatory proteins that bind to complementary receptors on the T cell surface; and (3) cell-cell adhesion molecules that allow T cells to bind to the APC for a sufficiently long time to become activated. system," Molecular Biology of the Cell, Alberts, B. et al., Garland Science, NY, (2002)).

[0139] T cells are subdivided into two distinct classes based on the cell surface receptors they express. Most T cells express T cell receptors (TCRs) composed of α and β chains. A small group of T cells express receptors composed of γ and δ chains. Among α / β T cells, there are two sub-lineages: those expressing the co-receptor molecule CD4 (CD4 + T cells); and expressing CD8 (CD8 + T cells). These cells differ in the way they recognize antigens and in their effectors and regulatory functions.

[0140] CD4+ T cells . CD4 + T cells are the primary regulatory cells of the immune system. Their regulatory functions depend on both the expression of cell-surface molecules, such as CD40L, which are induced when T cells are activated, and the wide range of cytokines they secrete upon activation. T cells also mediate important effector functions, some of which are determined by the pattern of cytokines they secrete. Cytokines can be directly toxic to target cells and can mobilize potent inflammatory mechanisms.

[0141] CD8+ T cells In addition, T cells, especially CD8 + T cells can develop into cytotoxic T-lymphocytes (CTLs) capable of efficiently eluting target cells expressing antigens recognized by CTLs (Paul, WE, "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, WE, Lippicott-Raven Publishers, Philadelphia, (1999)).

[0142] T cell receptors (TCRs) recognize complexes composed of peptides induced by the proteolysis of antigens bound to the differentiated grooves of Class II or Class I HLA proteins. CD4 + T cells recognize only peptide / class II complexes, whereas CD8 + T cells recognize peptide / class I complexes (Id.).

[0143] TCR ligands (i.e., peptide / HLA protein complexes) are generated within the APC. Generally, Class II MHC molecules bind to peptides derived from proteins uptaken by the APC via the process of endocytosis. These peptide-loaded Class II molecules are then expressed on the cell surface, where they are CD4 cells possessing a TCR capable of recognizing the expressed cell surface complex. + It is available for binding by T cells. Therefore, CD4 + T cells differentiate to react with antigens derived from extracellular sources (Id.).

[0144] In contrast, Class I HLA molecules are loaded primarily with peptides derived from internally synthesized proteins, such as viral proteins. These peptides are generated from cytoplasmic proteins by proteosome degradation and translocated into the rough endoplasmic reticulum. Generally composed of nine amino acids, these peptides bind within the Class I HLA molecule and express the appropriate CD8 receptor. + It moves to a cell surface that can be recognized by T cells. This is the T cell system, particularly CD8 + T cells provide the ability to detect cells that express proteins that are different from or produced in much greater quantities than those of cells of the rest of the organism (e.g., viral antigens) or mutant antigens (e.g., active oncogene products), even if these proteins are not expressed or secreted on the cell surface in their intact form (Id.).

[0145] T cells can also be classified based on their function as helper T cells; T cells involved in inducing cellular immunity; suppressor T cells; and cytotoxic T cells.

[0146] Helper T cells Helper T cells are T cells that stimulate B cells to elicit antibody responses against proteins and other T cell-dependent antigens. T cell-dependent antigens are immunogens in which individual epitopes appear only once or a limited number of times, causing them to be unable to cross-link to B cell membrane Ig or to cross-link inefficiently. B cells bind to the antigen via membrane Ig, and the complex undergoes endocytosis. Within the endosome and lysosome compartments, the antigen is fragmented into peptides by proteases, and one or more of the generated peptides are loaded into Class II HLA molecules passing through these vesicular compartments. The generated peptide / Class II HLA complex is then released onto the B cell surface membrane. T cells possessing receptors specific to the peptide / Class II molecule complex recognize this complex on the B cell surface. (Id.).

[0147] B-cell activation depends on both the binding of T cells via their TCRs and the interaction between CD40 on B cells and T-cell CD40L. T cells do not constitutively express CD40L. Rather, CD40L expression is induced as a result of interactions with congenital antigens recognized by the T cell's TCR and with APCs expressing either CD80 or CD86. CD80 / CD86 is generally expressed by activated but non-resting B cells, where helper interactions involving activated B cells and T cells can induce efficient antibody production. However, in many cases, the initial induction of CD40L on T cells depends on the recognition of that antigen on the surface of APCs that constitutively express CD80 / 86, such as DCs. These activated helper T cells can then efficiently interact with and assist B cells. Cross-linking of membrane Ig on B cells can generate active B-cell activation in synergy with CD40L / CD40 interactions, even if inefficient. In B-cell responses including proliferation, Ig secretion, and class switching of the expressed Ig class, subsequent events depend on or are enhanced by the action of T cell-derived cytokines (Id.).

[0148] CD4 + T cells are cells that primarily secrete the cytokines IL-4, IL-5, IL-6, and IL-10 (T H 2 cells) or cells that primarily produce IL-2, IFN-γ, and lymphotoxins (T H It tends to differentiate into 1 cell). T H While 2 cells are very effective in helping B cells develop into antibody-producing cells, T H 1. Cells are effective inducers of cellular immune responses that enhance the bactericidal activity of monocytes and macrophages, thereby consequently increasing the efficiency of leaching microorganisms from the intracellular endoplasmic reticulum compartment. HCD4 with 2 cell phenotypes (i.e., IL-4, IL-5, IL-6, and IL-10) + T cells are efficient helper cells, but T H 1 Cells also have the ability to become helpers (Id.).

[0149] T cell involvement in cellular immunity induction. T cells can also act to enhance the ability of monocytes and macrophages to destroy intracellular microorganisms. In particular, interferon-gamma (IFN-γ) produced by helper T cells enhances various mechanisms by which monocytes destroy intracellular bacteria and parasites, including the production of nitric oxide and the induction of tumor necrosis factor (TNF). H1 Since cells produce IFN-γ, they are effective in enhancing bactericidal action. In contrast, T H2 IL-4 and IL-10, two major cytokines produced by cells, block these activities (Id.).

[0150] Cytotoxic T lymphocytes. CD8 that recognizes peptides from proteins produced within target cells + T cells possess cytotoxic properties in that they induce the efflux of target cells. The mechanism of action for CTL-induced efflux involves the production of perforin by CTLs, a molecule capable of intercalating into the target cell membrane and promoting its efflux. Perforin-mediated efflux is enhanced by granzymes, a series of enzymes produced by activated CTLs. Many active CTLs also express large amounts of Fas ligands on their surfaces. The interaction between Fas on the surface of target cells and Fas ligands on the surface of CTLs initiates apoptosis in the target cells, leading to their death. CTL-mediated efflux appears to be a major mechanism of action for the destruction of virus-infected cells.

[0151] Regulatory T (Treg) cells. Immune homeostasis is maintained by a regulated balance between the initiation and downregulation of the immune response. The mechanisms of action of apoptosis and T cell energization (a resistance mechanism in which T cells are essentially functionally inactivated following antigen encounter) contribute to the downregulation of the immune response (Scwartz, RH Annu. Rev. Immunol., 21: 305-334(2003)). A third mechanism involves repressors or regulators CD4 + It is provided by the inhibition of activated T cell activity by T (Treg) cells (Reviewed in Kronenberg, M. et al. Nature, 435: 598-604 (2005)). IL-2 receptor alpha (IL-2Rα) chain (CD4 + CD25 + CD4 that constitutively expresses ) + Tregs are an energetic and repressive naturally occurring subset of T cells (Taams, LS et al. Eur. J. Immunol. 31: 1122-1131(2001)). CD4 + CD25 + Treg depletion causes systemic autoimmune disease in mice. Furthermore, the transfer of these Tregs prevents the onset of autoimmune disease. Human CD4, similar to its Muirline counterpart + CD25 + Tregs are produced in the thymus and have the ability to inhibit the proliferation of responder T cells through a cell-to-cell contact-dependent mechanism, the ability to produce IL-2, and In vitro It is characterized by the phenotype of the anergy. Human CD4 + CD25 + T cells are suppressed depending on the level of CD25 expression (CD25 high ) and non-inhibitory (CD25 low ) can be divided into cells. FOXP3, a member of the forkhead family of transcription factors, is Muirline and human CD4 + CD25 + It was shown to be expressed in Tregs and CD4+ CD25 + It appears to be a master gene that controls Treg development (Battaglia, M. et al. J. Immunol., 177: 8338-8347, (2006)).

[0152] T-memory cell. After recognizing and eradicating pathogens through an adaptive immune response, the majority of T cells (90-95%) undergo apoptosis, and the remaining cells become central memory T cells (T CM ), effector memory T cells (T EM ) and resident memory T cells (T RM Forms a pool of memory T cells designated as ) (Clark, RA Sci. Transl. Med., 7, 269rv1, (2015)).

[0153] Compared to standard T cells, these memory T cells are long-lived with distinct phenotypes, such as the expression of specific surface markers, the rapid generation of different cytokine profiles, the ability to perform direct effector cell functions, and unique homing distribution patterns. Memory T cells exhibit a rapid response upon re-exposure to their respective antigens to eliminate reinfection by attackers and thereby rapidly restore the balance of the immune system. Increasing evidence suggests that autoimmune memory T cells hinder most attempts to treat or cure autoimmune diseases (Id.).

[0154] Dendritic cells (DC)

[0155] DCs reside in tissues and play a crucial role in initiating and controlling the magnitude and quality of adaptive immune responses. Immature DCs act as watchdogs for potentially dangerous signals from cancer cells or microorganisms and possess potent phagocytic antigen capture capabilities. Upon maturation stimulation, immature DCs lose expression of adhesion molecules, undergo cytoskeletal reorganization, and migrate to draining lymph nodes. Mature DCs are specialized antigen-presenting cells and possess increased expression of MHC class II and co-stimulatory molecules on their cell surfaces. Innate immune responses detect typical pathogen molecules using pattern recognition receptors, which are germline receptors encoded to recognize a limited number of patterns. These receptors include Toll-like receptors, cell surface C-type lectin receptors, and intracellular nucleotide oligomerization domain (NOD)-like receptors (see discussion below; also see Alberts, DS, and LM Hess, editors. Fundamentals of Cancer Prevention. Springer Nature, 2019).

[0156] Monocytes (MO) and macrophages (MΦ)

[0157] Monocytes (MO), macrophages (MΦ), and tumor-associated macrophages (TAM) are part of the bone marrow family (a group of hematopoietic-derived cells). Monocytes are direct precursors of hematopoietic stem cell-derived macrophages. After being recruited into tumor tissue, they can differentiate into tumor-associated macrophages (TAMs), a cell population with high phenotypic and anti-tumor functional heterogeneity. They have been shown to support tumor initiation, local progression, and distant metastasis (Richards, David M, et al., "Monocytes and Macrophages in Cancer: Development and Functions." Cancer Microenvironment: Official Journal of the International Cancer Microenvironment Society, Springer Netherlands, Aug. 2013, www.ncbi.nlm.nih.gov / pmc / articles / PMC3). However, other studies have shown that TAMs also possess anti-tumor properties.

[0158] MO cells are primarily found in the bone marrow but are also found in the blood and spleen. Studies indicate that MO cells can also be generated extramedullary to hematopoiesis in the spleen and can increase under inflammatory conditions, such as the presence of cancer. MOs generated from hematopoietic stem cells (HSCs) undergo a series of differentiation processes and a commitment phase collectively known as monopoly. Monopoly is strictly regulated by microenvironmental signals, which control gene expression in developing cells and induce often irreversible phenotypic and functional changes associated with hematopoietic differentiation. While cytokines such as M-CSF, GM-CSF, and IL-3 are known to play a role in monopoly, other relatively unstudied factors, such as stromal cells or extracellular matrix (ECM) components, may also influence monopoly. (Id).

[0159] Different subsets of monocytes are generated from HSCs in the bone marrow through the exclusive progression to macrophage dendritic cell progeneration (MDP), which acts as a precursor for MO, macrophages (MΦ), and DCs, via distinct proliferation factor progeneration stages such as common myeloid progeneration (CMP) and granulocyte / macrophage progeneration (GMP). Once differentiated into MO, it becomes "classical" Ly6C high (Non-patrol) and "Non-classical" Ly6C low(Patrol) MOs: It should be noted that while they can be organized into two groups, the phenotypic or behavioral significance between these two subsets is not fully understood. The monocyte subset is mobilized from the bone marrow into the bloodstream, where it forms a local reservoir in the spleen; from the spleen, it can be re-mobilized in response to injury or inflammation. Under steady-state homeostasis, blood monocytes are recruited to other tissues, generating MO-derived MΦ and DCs involved in tissue development and the maintenance of homeostasis (ID). Conversely, in the presence of a tumor, MOs can generate populations of immunosuppressive TAMs and monocyte MDSCs in the tumor microenvironment, where they can promote tumor progression and immune evasion.

[0160] In tissues, MΦ supports homeostatic or trophic processes for tissue development / remodeling in tissue development or healing. The mechanisms of action involved in this support include phagocytosis, growth factor production, angiogenesis, and the degradation of ECM components. In response to immunogenic signals, such as TLR signaling or inflammatory cytokines, the functional characteristics of macrophages are polarized regarding processes required for immunity and pathogen defense. These include pathogen phagocytosis, release of cytotoxic reactive oxygen / reactive nitrogen species (RO / RNS), production of anti-inflammatory cytokines, and HLA class II-mediated antigen presentation (see id).

[0161] MΦ can be organized on a linear scale according to polarization state, namely from anti-inflammatory M1-MΦ (classically activated) to anti-inflammatory M2-MΦ (alternatively activated). TAMs are generated when tumor-derived factors are attracted and differentiate into MΦs. Similar to non-TAM MΦs, TAMs exhibit phenotypic and functional heterogeneity that varies depending on the type of tissue and tumor, the stage of tumor progression, and the location within the tumor tissue. For example, increased density of M2-like TAMs is a marker of poor prognosis in certain types of cancer (e.g., breast, cervical, and bladder cancer), whereas increased density of M1-like TAMs is a marker of better prognosis in other types of cancer (e.g., prostate, lung, and brain).

[0162] TAMs can influence almost every stage of tumor development and progression. Studies have shown that they possess a wide range of anti-tumor functions, including the generation of cytotoxic factors, phagocytosis of tumor cells (e.g., metastatic cells), and participation in cancer immuno-editing. (See Bingle L, Brown NJ, Lewis CE. The role of tumor-associated macrophages in tumor progression: implications for new anticancer therapies. J Pathol. (2002) 196: 254-265; also see O'Sullivan T, Saddawi-Konefka R, Vermi W, Koebel CM, Arthur C, White JM, Uppaluri R, Andrews DM, Ngiow SF, Teng MW, Smyth MJ, Schreiber RD, Bui JD. Cancer immunoediting by the innate immune system in the absence of adaptive immunity. J Exp Med. (2012) 209: 1869-1882).

[0163] Studies have shown that TAMs can be biased toward anti-tumor functions. For example, activation of the CD40 pathway has been reported to program TAMs to upregulate the expression of MHC class II and the co-stimulatory molecule CD86 accumulation in tumor tissues, leading to TAM-mediated elution of tumor cells. IL-12 and TNF-α-mediated treatment has also been reported to program TAMs to display their anti-tumor effector functions. (See Watkins SK, Egilmez NK, Suttles J, Stout RD. IL-12 rapidly alters the functional profile of tumor-associated and tumor-infiltrating macrophages in vitro and in vivo. J Immunol. (2007) 178:1357-1362). CD47 acts as an anti-phagocyte signaler by binding to SIRPα, a protein expressed on MΦ and DCs. It has been found that blocking CD47 activity or SIRPα access results in MΦ-dependent phagocytosis of tumor cells. (Chao MP, Weissman IL, Majeti R. The CD47-SIRPalpha pathway in cancer immune evasion and potential therapeutic implications. Curr Opin Immunol. (2012) 24: 225-232).

[0164] MO, MΦ, DC, and related cells are currently identified by the phenotypic markers shown in Table 1.

[0165] [Table 1]. MO, MΦ, and DC phenotypes.

[0166]

[0167] The terms “Major Histocompatibility Complex (MHC), MHC-like molecule,” and “HLA” are used interchangeably herein to refer to cell-surface molecules that denote molecular fractions known as epitopes or antigens and mediate interactions between leukocytes and other leukocytes or somatic cells. MHC is encoded by a large group of genes and can be organized into three subgroups: Class I, Class II, and Class III. In humans, the MHC gene complex is called HLA (“Human Leukocyte Antigen”); in mice, it is called H-2 (in the case of “Histocompatibility”). Both species possess three major MHC Class I genes, called HLA-A, HLA-B, and HLA-C in humans, and H2-K, H2-D, and H2-L in mice. These encode the α chain of the respective MHC Class I protein. Another subunit of the MHC Class I molecule is β2-microglobulin. Class II legons include genes for the α and β chains (designated A and B) of the MHC class II molecules HLA-DR, HLA-DP, and HLA-DQ in humans. Additionally, the MHC class II region contains genes for the TAP1:TAP2 peptide transporter, the PSMB (or LMP) gene encoding the proteasome subunit, genes encoding the DMα and BMβ chains (DMA and DMB), genes encoding the α and β chains of the DO molecule (DOA and DOB, respectively), and the gene encoding tapacin (TAPBP). Class II genes encode various other proteins that function in immunity. The DMA and DMB genes, which allow for the HLA-DM molecule subunits that catalyze peptide binding to MHC class II molecules, are associated with MHC class II genes, as are the DOA and DOB genes encoding the regulatory HLA-DO molecule subunits. Janeways Immunobiology. 9th ed., GS, Garland Science, Taylor& Francis Group, 2017.pps. 232-233.

[0168] MHC-like molecules are not encoded by the same gene groups as true MHC, but they possess the same folding and overall structure as MHC, specifically MHC class I molecules, and thus possess similar biological functions such as antigen presentation. The CD1 family of molecules is an example of MHC-like molecules. It consists of two groups based on amino acid homology: Group 1, which includes CD1a, b, and c; and Group 2, which consists of CD1d. Group 1 CD1s can present antigens to various T cells, whereas CD1d primarily presents antigens to NKT cells. (Brutkiewicz. "CD1d Ligands: The Good, the Bad, and the Ugly." The Journal of Immunology (2006) 177 (2) 769-775). Although CD1d is structurally similar to MHC class I molecules, it moves through endosomes in the exogenous antigen presentation pathway. The binding groove of the CD1d molecule binds to the lipid tail of glycolipid antigens, whereas the carbohydrate head group of the antigen protrudes out of the groove for recognition by the TCR of NKT cells. (Wah, MakTak, et al. "Chapter 11: NK, γδ T and NKT Cells." Primer to the Immune Response. Elsevier, 2014).

[0169] CD1d presents lipid antigens and requires the presence of specific mechanisms of action to induce the uptake of these molecules by APCs and subsequent loading onto CD1d molecules. Lipid transport proteins, such as apolipoprotein E and fatty acid amide hydrolase (FAAH), have been shown to enhance the presentation of specific antigens by CD1d. Loading efficiency can be enhanced by specific proteins, such as saponins and microsomal triglyceride transport proteins present in the cellular endosome and lysosome compartments, by facilitating lipid antigen exchange. Similar to MHC antigens, lipid antigens may also be processed by lysosomal enzymes to produce active compounds, as demonstrated in the case of CD1d for synthetic antigens, microbial antigens, and self-antigens. Giradi and Zajonc (2012). "Molecular basis of lipid antigen presentation by CD1d and recognition by natural killer T cells." Immunol Rev. 250(1): 167-179.

[0170] While MHC class I-like molecules are non-classical MHC type molecules, those including CD1a, CD1b, CD1c, CD1e, and Cd1d (including MR1) are also expressed on APCs and can activate various subsets of T cells. Kumar and Delovitch (2014) "Different subsets of natural killer T cells may vary in their roles in health and disease." Immunology 142: 321-336. Other non-classical histocompatibility molecules include MR1, which activates MAIT cells.

[0171] The terms “marker” or “cell surface marker” are used interchangeably herein to refer to an epitope or antigenic determinant found on the surface of a specific type of cell. Cell surface markers can facilitate the characterization, identification, and ultimately isolation of cell types. Cell classification techniques are based on cell biomarkers in which cell surface marker(s) can be used for positive or negative selection, i.e., inclusion or exclusion from a cell population.

[0172] The terms “mixed lymphocyte reaction” or “MLR” are used interchangeably herein to refer to an in vitro cellular immunoassay occurring between two allogeneic lymphocyte populations. In a classic mixed lymphocyte reaction (MLR), a suspension of responder T cells is cultured with allogeneic (donor) stimulator cells. Foreign MHC class I or class II molecules expressed by the allogeneic stimulator cells serve as activating stimuli for the responding T lymphocytes. The proliferation of the responding T lymphocytes is then measured. Additionally, the stimulator cell population containing T-cells will replicate in the presence of responder cells (bidirectional mixed lymphocyte reaction). In the case of a unidirectional mixed lymphocyte reaction, the stimulator cells are prevented from replicating by, for example, irradiation or treatment with mitomycin C to prevent cell replication.

[0173] The terms “mixed lymphocyte tumor response” or “MLTR” are used interchangeably herein to refer to a response similar to a mixed lymphocyte response in which allogeneic tumor cells are used rather than allogeneic lymphocytes to stimulate the response. An MLTR method comprises contacting a mixed lymphocyte population with allogeneic tumor cells. One or more of the following are measured: cell proliferation of lymphocytes, differentiation of lymphocyte cell subsets, cytokine release profiles of lymphocytes, and tumor cell death.

[0174] As used in this specification, the term "modify" refers to a change in form or quality.

[0175] As used herein, the term “modify” means to regulate, change, adapt, or adjust by a specific scale or rate. Such regulation may be any change, including undetectable changes. In the context of a series of apoptotic cell types such as NKCs, CTLs, and NKTs, the terms “modified” or “modified” as used herein mean to alter the form or characteristics of a cell type through one or more recombinant DNA technologies such that the immunostimulatory or immunosuppressive effect of each modified series of apoptotic cells is reproducibly different from that of each parent series of apoptotic cells.

[0176] As used herein, the term “mononucleus” refers to cells having a single circular nucleus, such as lymphocytes (e.g., T cells, B cells, NK cells), monocytes, and macrophages.

[0177] As used herein, the terms “myeloid suppressor cell,” “myeloid-derived suppressor cell,” or “MDSC” refer to a heterogeneous population of cells characterized by a myeloid origin, an immature state, and the ability to potently suppress T cell responses. These cells regulate immune responses and tissue repair in healthy individuals, and the population expands rapidly during inflammation.

[0178] The term “nucleic acid” is used herein to refer to deoxyribonucleotides or ribonucleotide polymers in a single- or double-stranded form, and, unless otherwise limited, encompasses known analogs having the essential properties of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids).

[0179] The term “nucleotide” is used herein to refer to a chemical compound composed of a heterocyclic base, a sugar, and one or more phosphate groups. In the most common nucleotides, the base is a derivative of a purine or pyrimidine, and the sugar is pentose deoxyribose or ribose. Nucleotides are monomers of nucleic acids in which three or more are joined together to form nucleic acids. Nucleotides are the structural units of RNA, DNA, and various cofactors, including but not limited to CoA, FAD, DMN, NAD, and NADP. Purines include adenine (A) and guanine (G); pyrimidines include cytosine (C), thymine (T), and uracil (U).

[0180] As used herein, the term “open reading frame” refers to a sequence of nucleotides in a DNA molecule that may encode a peptide or protein: it begins with a start triplet (ATG), is followed by a sequence of triplet symbols, each encoding an amino acid, and ends with a stop triplet (TAA, TAG, or TGA).

[0181] The phrase “operably linked” refers to (1) a first sequence(s) or domain located sufficiently close to a second sequence(s) or domain so that the first sequence(s) or domain can influence the second sequence(s) or domain or a region under the control of the second sequence(s) or domain; and (2) a functional link between a promoter and a second sequence, wherein the promoter sequence initiates and mediates the transcription of a DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are adjacent and are in the same read frame as is necessary to link two protein-coding regions. According to some embodiments, the phrase “operably linked” refers to a link in which two or more protein domains or polypeptides are ligated or combined via recombinant DNA technology or chemical reactions so that each protein domain or polypeptide of the resulting fusion protein retains its original function.

[0182] As used herein, the term "total survival" (OS) refers to the length of time from the date of diagnosis of the disease or the start of treatment during which a patient diagnosed with the disease is still alive.

[0183] As used herein, the term "perisecretory signaling" refers to short-range cell-to-cell communication through secreted signaling molecules acting on adjacent cells.

[0184] As used herein, the term “parenteral” and other grammatical forms thereof refer to the administration of a substance occurring in the body other than the mouth or digestive tract. For example, as used herein, the term “parenteral” refers to introduction into the body by injection (i.e., administration by injection), including, for example, subcutaneous (i.e., injection under the skin); intramuscular (i.e., intramuscular injection); intravenous (i.e., intravenous injection); intrathecal (i.e., injection into the space between the spinal cord or the subarachnoid space of the brain); or infusion techniques.

[0185] As used herein, the term "passive immunization" refers to the generation of passive immunity, which means immunity acquired naturally, from the transfer of antibodies from mother to fetus, or through intentional inoculation (artificial passive immunity). Passive immunity can be induced by natural or artificial mechanisms of action. When antibodies are transferred, passive immunity against the specific antibody transferred is specific. Passive cell-mediated immunity is generated by the transfer of living lymphocytes from an immune cell source and is sometimes referred to as progenitor or acquired immunity.

[0186] The terms "peripheral blood mononuclear cell" or "PBMC" are used interchangeably herein to refer to mononuclear cells derived from peripheral blood.

[0187] As used herein, the term “pharmaceutical composition” refers to a composition used to prevent, reduce in intensity, cure, or otherwise treat a target condition, syndrome, disorder, or disease. The terms “formulation” and “composition” are used interchangeably herein to refer to the product of the described invention comprising all active and inactive ingredients.

[0188] As used herein, the term “pharmaceuticalally acceptable carrier” refers to any substantially non-toxic carrier that is ordinarily available for administration of the activator of the described invention, in which the activator is maintained in a stable and bioavailable state. The pharmaceutically acceptable carrier must be of sufficiently high purity and sufficiently low toxicity to be suitable for administration to the mammal being treated. Furthermore, it must maintain the stability and bioavailability of the activator. The pharmaceutically acceptable carrier may be liquid or solid and is selected with in mind a planned method of administration to provide the desired volume, consistency, etc., when combined with the activator and other components of a given composition.

[0189] As used herein, the term “pharmaceuticalally acceptable salt” refers to salts that, within the scope of sound medical judgment, are suitable for use in contact with tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., and correspond to a reasonable benefit / risk ratio. When used in medicine, the salt must be pharmaceutically acceptable, but the pharmaceutically acceptable salt may be prepared by conveniently using non-pharmaceutically acceptable salts. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric acid, hydrobromide, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, p-toluenesulfonic acid, tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Additionally, such salts may be prepared as alkali metal or alkaline earth salts, such as sodium, potassium, or calcium salts of carboxylic acid groups. "Pharmaceuticalally acceptable salts" means salts that are suitable for use in contact with human and lower animal tissues without excessive toxicity, irritation, allergic reactions, etc., within the scope of sound medical judgment, and correspond to a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, PH Stahl et al. describe pharmaceutically acceptable salts in detail in the "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley VCH, Zurich, Switzerland: 2002). Salts may be prepared separately during the final isolation and purification of the compounds described in this invention, either in situ or by reacting free base functional groups with a suitable organic acid.Representative acid addition salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate (isethionate), lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesulfonate, and It includes, but is not limited to, undecanoates. Additionally, the basic nitrogen-containing group may be quaternized to preparations such as lower alkyl halides, e.g., methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dialkyl sulfates, such as dimethyl, diethyl, dibutyl, and diamyl sulfates; long-chain halides, e.g., decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; and arylalkyl halides, such as benzyl and phenethyl bromides. This yields a water or oil-soluble or dispersible product. Examples of acids that may be used to form pharmaceutically acceptable acid addition salts include inorganic acids such as hydrochloric acid, hydrobromide, sulfuric acid, and phosphoric acid, and organic acids such as oxalic acid, maleic acid, succinic acid, and citric acid. Basic addition salts can be prepared in situ during the final isolation and purification of the compounds described in the invention by reacting the carboxylic acid-containing moiety with a suitable base, such as a hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation, or with ammonia or an organic primary, secondary, or tertiary amine.Pharmaceutically acceptable salts include, but are not limited to, cations based on alkali metals or alkaline earth metals such as lithium, sodium, potassium, calcium, magnesium, and aluminum salts, and non-toxic quaternary ammonia and amine cations including ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, etc. Other representative organic amines useful for forming base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, etc. Pharmaceutically acceptable salts may also be obtained using standard procedures widely known in the art by reacting a sufficiently basic compound, for example, an amine, with a suitable acid that provides a physiologically acceptable anion. Alkali metal (e.g., sodium, potassium, or lithium) or alkaline earth metal (e.g., calcium or magnesium) salts of carboxylic acids may also be prepared.

[0190] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. The terms apply to naturally occurring amino acid polymers as well as to amino acid polymers in which one or more amino acid residues are synthetic chemical analogs of corresponding naturally occurring amino acids. An essential characteristic of these naturally occurring amino acid analogs is that when incorporated into a protein, the protein responds specifically to an antibody composed entirely of naturally occurring amino acids, although the protein is derived for the same protein. The terms “polypeptide,” “peptide,” and “protein” also include modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation, hydroxylation, and ADP-ribosylation of glutamic acid residues. As is well known and mentioned above, it will be understood that polypeptides may not be completely linear. For example, polypeptides may be branched as a result of ubiquitination and may be circular with or without branching as a result of post-translational events, including generally natural processing events and events caused by human manipulation that do not occur naturally. Circular, branched, and branched circular polypeptides may also be synthesized by non-translational natural processes and entirely synthetic methods. According to some embodiments, the peptide is of any length or size.

[0191] The terms "protein domain" and "domain" are used interchangeably to refer to parts of a protein that have their own three-dimensional structure. Large proteins generally consist of multiple domains connected to each other through flexible regions of polypeptide chains.

[0192] The following terms are used herein to describe sequence relationships between two or more nucleic acids or polynucleotides: (a) “reference sequence,” (b) “comparison window,” (c) “sequence identity,” (d) “percentage of sequence identity,” and (e) “substantial identity.” (a) The term “reference sequence” refers to a sequence used as the basis for sequence comparison. The reference sequence may be a subset or the whole of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or a complete cDNA or gene sequence. (b) The term “comparison window” refers to an adjacent and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence may be compared with the reference sequence, and the portion of the polynucleotide sequence in the comparison window may include additions or deletions (i.e., gaps) compared with the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 consecutive nucleotide lengths, optionally at least 30 consecutive nucleotide lengths, at least 40 consecutive nucleotide lengths, at least 50 consecutive nucleotide lengths, at least 100 consecutive nucleotide lengths, or longer. Those skilled in the art understand that a gap penalty is typically introduced and subtracted from the number of matches to avoid high similarity to a reference sequence due to the inclusion of gaps in the polynucleotide sequence. Alignment methods for comparison are well known in the art. The optimal alignment of sequences for comparison is Smith and Waterman, Adv. Appl. Math . Local homology algorithm of 2:482 (1981); Needleman and Wunsch , J. Mol. Biol . 48:443 (1970)'s homology sorting algorithm; Pearson and Lipman, Proc. Natl. Acad. Sci. A method for testing similarity of 85:2444 (1988); CLUSTAL in the PC / Gene program by Intelligenetics, Mountain View, California; can be performed by computerized implementations of these algorithms including, but not limited to, GAP, BESTFIT, BLAST, FASTA, and TFASTA by the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Doctor Madison, Wisconsin, USA; and the CLUSTAL program is by Higgins and Sharp, Gene 73:237-244 (1988); Higgins and Sharp; CABIOS 5:151-153 (1989); Corpet, et al., Nucleic Acids Research 16:10881-90 (1988); Huang, et al., Computer Applications in the Biosciences, 8:155-65 (1992), and Pearson, et al., Methods in Molecular Biology This is well described by , 24:307-331 (1994). The BLAST family of programs that can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences. Current Protocols in Molecular BiologySee , Chapter 19, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995). Unless otherwise specified, sequence identity / similarity values ​​provided herein refer to values ​​obtained using the BLAST 2.0 program suite using basic parameters. Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Software for performing BLAST analysis is publicly available, for example, through the National Center for Biotechnology-Information. This algorithm involves identifying high-scoring sequence pairs (HSPs) by first identifying short words of length W in query sequences that match or satisfy some positive-value threshold score T when aligned with words of the same length in database sequences. T is referred to as the neighbor word score threshold (see Altschul et al., cited above). These initial neighbor word hits serve as seeds to initiate a search and find longer HSPs containing them. Then, word hits are extended in both directions along each sequence as the cumulative alignment score can increase. The cumulative score is calculated for nucleotide sequences using parameters M (reward score for pairs of matching residues; always >0) and N (penalty score for non-matching residues, always <0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. The extension of word hits in each direction is halted in the following cases: when the accumulated alignment score falls by a quantity X from its maximum achieved value; when the accumulated score becomes zero or less due to the accumulation of one or more negative-scoring residue alignments; or when the end of any sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of alignment. The BLASTN program (for nucleotide sequences) by default uses a word length (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program by default uses a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USASee 89:10915). In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of similarity between two sequences (e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA See 90:5873-5787 (1993). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences occurs by chance. BLAST search assumes that proteins can be modeled as random sequences. However, many real proteins contain regions of non-random sequences that may be homopolymer tracks, short-period repeats, or regions rich in one or more amino acids. These low-complexity regions can align between unrelated proteins even if other regions of the protein are not completely similar. To reduce these low-complexity alignments, multiple low-complexity filter programs can be used. For example, SEG (Wooten and Federhen, Comput. Chem ., 17:149-163 (1993)) and XNU (Claverie and States, Comput. Chem(c) In the context of two nucleic acid or polypeptide sequences, the terms “sequence identity” or “identity” are used herein to refer to residues in the same two sequences when aligned for maximum match across a specified comparison window. When the percentage of sequence identity is used in relation to proteins, it is recognized that non-identical residue positions are often different due to conservative amino acid substitutions, that is, where an amino acid residue is substituted with another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity) and thus does not alter the functional properties of the molecule. Where sequences differ due to conservative substitutions, the percentage sequence identity may be up-adjusted to correct for the conservative nature of the substitution. Sequences that differ due to such conservative substitutions are referred to as having “sequence similarity” or “similarity.” Means for making this adjustment are well known to those skilled in the art. Typically, this involves increasing percentage sequence identity by scoring partially conservative substitutions rather than complete mismatches. Thus, for example, if the same amino acid is given a score of 1 and a non-conservative substitution is given a score of 0, conservative substitutions are given a score between 0 and 1. Scoring of conservative substitutions is implemented, for example, in the program PC / GENE (Intelligenetics, Mountain View, California), for example by Meyers and Miller, Computer Applic. Biol. Sci(d) The term “percentage of sequence identity” as used herein means a value determined by comparing two optimally aligned sequences for a comparison window, wherein a portion of the polynucleotide sequence in the comparison window may include additions or deletions (i.e., gaps) compared with a reference sequence (without additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to calculate the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to calculate the percentage of sequence identity. (e) The term “substantial identity” of a polynucleotide sequence means that the polynucleotide comprises a sequence having at least 60% sequence identity, at least 70% sequence identity, at least 80% sequence identity, at least 90% sequence identity, and at least 95% sequence identity when compared to a reference sequence using one of the alignment programs described using standard parameters. Those skilled in the art will recognize that these values ​​may be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, read frame localization, etc. For these purposes, substantial identity of amino acid sequences generally means at least 60%, or at least 70%, at least 80%, at least 90%, or at least 95% sequence identity. Another indication that nucleotide sequences are substantially identical is when two molecules hybridize with each other under strict conditions. However, nucleic acids that do not hybridize with each other under strict conditions are still substantially identical if the polypeptides they encode are substantially identical. This can occur, for example, when a copy of nucleic acid is generated using the maximum codon degeneracy allowed by the genetic code.One indication that two nucleic acid sequences are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid. Mutations can also be made for the nucleotide sequences of the protein by referencing the genetic code, including considering codon degeneracy.

[0193] As used herein, the term "prime" (or "priming") refers to a process of increasing sensitivity. When used in an immunological sense, it refers to the process by which T cell and B cell precursors encounter a specific antigen. As used herein, the term "unprimed cell" (also referred to as virgin, naive, or inexperienced cell) refers to T cells and B cells that generate antigen receptors of a specific specificity (TCR for T cells, BCR for B cells) but have never encountered an antigen. For example, before helper T cells and B cells can interact to produce a specific antibody, antigen-specific T cell precursors must be primed. Priming involves several steps: uptake of an antigen bound to a class II MHC molecule by an antigen-presenting cell, processing and cell surface expression; recycling and antigen-specific capture of helper T cell precursors in lymphoid tissue; and T cell proliferation and differentiation (Janeway, CA, Jr., Semin. Immunol.(1989) 1(1): 13-20). Helper T cells express CD4, but not all CD4 T cells are helper cells (Id.). The signal required for the clonal expansion of helper T cells is different from the signal required for other CD4 T cells. The important antigen-presenting cell for helper T cell priming appears to be a macrophage; and the important second signal for helper T cell growth is the macrophage product interleukin 1 (IL-1) (Id.). When a primed T cell and / or B cell receives a second, co-stimulatory signal, it becomes an activated T cell or B cell.

[0194] As used herein, the terms "progression-free survival" or "PFS" refer to the period during and after treatment for a disease, such as an autoimmune disease, during which a patient lives with the disease but does not experience further deterioration. Measuring progression-free survival in clinical trials is one method of determining how well a new treatment works.

[0195] As used herein, the term "purification" and its various grammatical forms refer to the process of isolating or removing foreign, external, or unpleasant elements. Accordingly, the term "purified substance" refers to a substance free from foreign, external, or unpleasant elements.

[0196] The terms "reporter gene" (“reporter”) or "test marker" refer to genes and / or peptides that can be detected or easily identified and measured. The expression of a reporter can be measured at the RNA level or the protein level. Gene products that can be detected in experimental test protocols include, but are not limited to, marker enzymes, antigens, amino acid sequence markers, cell phenotype markers, nucleic acid sequence markers, etc. Researchers may attach reporter genes to other genes of interest in cell cultures, bacteria, animals, or plants. For example, some reporters are selectable markers or characterize organisms expressing them, enabling the organism to be easily identified and tested. To introduce a reporter gene into an organism, researchers may place the reporter gene and the gene of interest in the same DNA construct so that they are inserted into the cell or organism. In the case of cultured bacteria or eukaryotic cells, this may be in the form of a plasmid. Commonly used reporter genes may include, but are not limited to, fluorescent proteins, luciferase, beta-galactosidase, and selectable markers such as chloramphenicol and caromycin.

[0197] As used herein, the term “a series of apoptotic cells” refers to a population of cells that exhibit the ability to kill multiple tumor or pathogen-infected cells while exhibiting resistance to such apoptotic action. There are various types of cells exhibiting this effector function, e.g., NK cells, NKT cells, LAK cells, CIK cells, MAIT cells, CD8+ CTLs, and CD4+ CTLs. This apoptotic effector function may be direct through cell lysis or cytotoxic activity, or indirect through the immunomodulation of other cells and proteins that target pathogenic and cancerous cells.

[0198] Effector series of dead cells

[0199] A series of apoptotic cells in the immune system can provide rapid immunity against pathogens through a series of apoptotic actions. There are various types of cells that exhibit this effector function. According to some embodiments, a series of apoptotic cells includes natural killer (NK) cells, natural apoptotic T (NKT) cells, CD8+ cell-lysing T lymphocytes (CTLs), and CD4+ CTLs. A series of apoptotic functions is defined as the ability to kill multiple tumor or pathogen-infected cells while exhibiting resistance to such apoptotic actions. A series of apoptotic effector functions may be direct through cell-lysing or cytotoxic activity, or indirect through the immunomodulation of other cells and proteins that target pathogenic and cancerous cells.

[0200] Although activation pathways may differ, a series of apoptotic cells can directly kill target cells through perforin / granzyme or granulicin mechanisms. The first step of the cytotoxic process is the recognition of target cells, which can be specific (in the case of CTLs) or non-specific (in the case of NK and NKTs). Subsequently, elutional synapses are formed between the series of apoptotic cells and the target cells. Adhesion molecules to the target cells (e.g., integrin LFA-1 containing its ligand ICAM-1 or ICAM-2) result in the polarization of cytotoxic granules toward the immunological synapses. When the Ig-superfamily receptor DNAM-1 (CD226), expressed by numerous immune cells such as NK, T, and B cells, associates with adhesion molecules on the surface of the series of apoptotic cells, it becomes phosphorylated and possesses the potential to transmit activation signals. Ligands of DNAM-1, such as PVR (CD155), a member of the nectin family of proteins, and Nectin-2 (CD11), a member of the nectin-like family of proteins, are frequently expressed by tumor cells. When a series of apoptotic cells are activated, cytotoxic granules containing perforin and granzyme, particularly granzyme B, are released. (Marcus, Assaf, et al., "Recognition of Tumors by the Innate Immune System and Natural Killer Cells.") Advances in Immunology , US National Library of Medicine, 2014, www.ncbi.nlm.nih.gov / pmc / articles / PMC4228931 / (see reference).

[0201] Perforin can polymerize, bind to target cells, and form channels on target cell membranes with the help of Ca2+ ions. Granzyme is a serine protease that enzymatically activates the caspase cascade that induces apoptosis. Perforin binds via membrane phospholipids, and phosphatidylcholine, which binds to Ca2+, increases perforin's affinity for the target cell membrane. The pores formed by perforin disrupt the cell membrane, allowing for the free influx and efflux of ions and polypeptides, and additionally permitting the delivery of granzyme molecules. Cell disruption and the delivery of cytotoxic granzyme ultimately induce the activation of anti-apoptotic pathways and DNA degradation, leading to cell death. This mechanism of action depends not only on the expression of perforin / granzyme but also on numerous other factors, such as temperature, pH, and calcium concentration. (See Lopez, Jamie A., et al., “Perforin Forms Transient Pores on the Target Cell Plasma Membrane to Facilitate Rapid Access of Granzymes during Killer Cell Attack.” Blood Journal, American Society of Hematology, 4 Apr. 2013, www.bloodjournal.org / content / 121 / 14 / 2659; also see Murphy, Kenneth M., et al., “T-Cell Mediated Immunity.” Janeways Immunobiology . 9th ed., GS, Garland Science, Taylor& Francis Group, 2017. pps. 387-395).

[0202] A series of apoptotic cells can also induce apoptosis via the apoptotic receptor / ligand pathway. For example, some series of apoptotic cells express a Fas ligand (FasL) on their cell membranes. When FasL comes into contact with and binds to Fas present on the membrane of a target cell, Fas ligates, causing the activation of caspases and inducing apoptosis in the target cell. (Paul, WE, "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, WE, Lippicott-Raven Publishers, Philadelphia, (1999)).

[0203] The Tumor Necrosis Factor (TNF)-associated Apoptosis-Inducing Ligand (TRAIL) receptor / TRAIL mechanism of action is another death receptor / ligand pathway. TRAIL is a transmembrane protein expressed in a wide range of apoptotic cell types that induce apoptotic death in various tumor cells but not in most normal cells. When the TRAIL ligand binds to either TRAIL-R1 or TRAIL-R2, the receptor is trimmed, and the Apoptosis-Inducing Signaling Complex (DISC) is assembled on the target cell. (Falschlehner, Christina, et al. "Following TRAIL's Path in the Immune System." Immunology, Blackwell Science Inc., June 2009, www.ncbi.nlm.nih.gov / pmc / articles / PMC2691779 / ).

[0204] Both Fas / FasL and TRAIL-R / TRAIL proceed as follows: The adapter molecule, the Fas-associated death domain (FADD), is translocated to DISC, where it interacts with the receptor's intracellular death domain. FADD recruits procaspases 8 and 10 to DISC via its secondary functional domain, the death effector domain (DED), where they are autocatalytically activated and thus initiate a caspase-dependent signaling cascade, ultimately leading to apoptosis. Id.

[0205] Many series of apoptotic cells exert immunomodulatory effects through the secretion of cytokines and chemokines that regulate the effector functions of surrounding cells, such as interferon-γ (IFN-γ), granulocyte macrophage colony-stimulating factor (GM-CSF), colony-stimulating factor 1 (CSF-1), tumor necrosis factor α (TNFα), transforming growth factor (TGFβ), interleukin (IL-3, IL-5, IL-10, IL-13), and chemokines (CCL1, 2, 3, 4) and (CXCL8). For example, several series of apoptotic cells secrete anti-inflammatory cytokines. Target cells can be sensitized to TRAIL-mediated apoptosis by the presence of inflammatory cytokines such as TNF-α and IFN-γ. While these cytokines can enhance apoptosis indirectly, they induce the generation of nitric oxide and other free radicals or disrupt apoptotic pathways within tumor cells. It can also be enhanced through a direct, contact-independent cytotoxic mechanism of action. IFN-γ has also been shown to activate macrophages and recruit them as sites of attack for both effector cell function and APCs. IFN-γ acts synergistically with TNF-α or TNF-β to kill certain target cells through its interaction with TNF receptor I (TNFR-I). (Ito and Seishima (2010), "Regulation of Induction and Function of Cytotoxic T Lymphocytes by Natural Killer T Cell." J Biomed Biotechnol, Art. ID. 641757).

[0206] There are several series of apoptotic cell types, such as various types of NKTs and NKs, that cannot be easily classified as a single arm of the immune system. These cell types include B-1 cells, specific subsets of marginal zone (MZ) B cells and γδ T cells, CD8αα-expressing T cells in the gut, cytokine-induced apoptotic cells, and MAIT cells. Each of these cell types expresses either a B cell receptor or a T cell receptor (TCR), which is an antigen-specific receptor. This is generated by VDJ recombination, a process in which T cells and B cells randomly assemble different gene segments known as variable (V), diversity (D), and linkage (J) genes to create unique antigen receptors capable of collectively recognizing many different types of molecules. Because the repertoire of specificity for these receptors is strongly restricted, these cells respond to antigens of limited diversity.

[0207] Receptors expressed by various types of NKTs and NKs bear similarities to pattern recognition receptors expressed by cells of the innate immune system. Cells of the T lymphocyte lineage further exhibit unique characteristics such as the inability to develop immunological memory, rapid induction of effector functions, and a tendency toward autoreactions. Despite sharing the T lymphocyte lineage with conventional T cells, these cells exhibit distinctly NK-like and T-cell-like characteristics, such as the ability to produce specific cytokines, the ability to activate other immune cells (e.g., conventional T cells, NKs, and / or DCs), and the ability to induce apoptosis through cytotoxic activity and / or expression, as well as the ability to interact with apoptosis-inducing effector molecules such as Fas ligands (FasL / Fas) and TNF-associated apoptosis-inducing ligands (TRAIL). (Ito and Seishima (2010), "Regulation of Induction and Function of Cytotoxic T Lymphocytes by Natural Killer T Cell." J Biomed Biotechnol, Art. ID. 641757). Other cells exhibiting similar effector functions, including CTLs, which share a series of apoptotic abilities with NKTs and NKCs.

[0208] Natural death (NK) cells

[0209] Natural Killer (NK) cells are cytolysable granulocytes found in humans and other mammals. They are characterized by their innate ability to lyse even in the absence of prior immunity to a target. (Seaman (2000) "Natural Killer Cells and Natural Killer T Cells." Arthritis & Rheumatism 43(6): 1204-1217).

[0210] NK cells have the form of activated cytotoxic T cells in that they are typically large with expanded cytoplasm containing granules used for cytotoxicity. NK cells can currently be identified by various surface receptors such as those shown in Table 2.

[0211] [Table 2]. Naturally dying cells

[0212]

[0213]

[0214]

[0215]

[0216] Specific NK cells will typically express two to four inhibitory receptors in addition to a series of activating receptors, and various combinations of inhibitory and activating receptors result in significant heterogeneity within the NK population. For this reason, NKs are considered to have the ability to respond to various stimuli and participate in diverse immune responses under different pathological conditions. (Mandal and Viswanathan (2015). "Natural killer cells: In health and disease." Hematol. Oncol. Stem Cell The. 8(2): 47-55).

[0217] NK cells, similar to B cells and myeloid-derived cells, primarily originate in the bone marrow. They have also been found to originate in lymph nodes and the liver. They can be generated from hematopoietic stem cells (HSCs) that demonstrate commitment to the NK lineage, thereby producing NK precursors (NKPs), which ultimately mature into NKs under the influence of specific transcription factors. Transcriptional, lytic, and membrane factors associated with NK development include Ets-1, Id2, Ikaros, and PU.1 during the generational stage; Gata-3 and IRF-2 during the maturation of immature NKs; and CEBP-γ, MEF, and MITF during the functional differentiation of mature NKs. The cytokine interleukin 15 (IL-15) has been shown to be essential for NK developmental homeostasis and survival. The cytokine interleukin-2 (IL-2), a peptide derived from T cells, is associated with the lytic and functional maturation of NK cells. (Id.).

[0218] NK cells are typically found circulating in peripheral blood until they become activated upon infiltrating most tissues containing pathogen-infected or malignant cells. They account for 10% of all cells in the total peripheral blood mononuclear cell (PBMC) population of circulating human lymphocytes. NK cells found in secondary lymphoid tissues, such as tonsils, lymph nodes, and the spleen, differ from peripheral blood NK cells in that lymphoid NK cells are activated by DCs and secrete specific cytokines, such as interferon, to stimulate a more efficient apoptotic response by T cells. (Id.).

[0219] NK stimulation and effector functions depend on the integration of signals derived from its various receptors. Through its cytotoxic function, NK can recognize and kill virus-infected cells and neoplastic cells.

[0220] Furthermore, NK cells perform an immunomodulatory role by stimulating the production of cytokines. In this manner, NK cells have the ability to regulate the activity of other cells, particularly those of the immune system. The pattern of cytokines released by NK induction varies depending on the stimulation. Therefore, NK cells, like T cells, differentiate into distinct functional subsets with different effects on adaptive immunity.

[0221] The presence of IFN-γ and other functional immunostimulatory factors, such as IL-2 and IL-12, on NK cells can induce the activation and expansion of NK cells into lymphokine-activated apoptotic (LAK) cells, which can be upgraded to cytokine-induced apoptotic cells (CIKs), which are CD3-, CD56-positive, non-major histocompatibility complex (MHC)-restricted, natural death (NK)-like T lymphocytes. LAK cells upregulate effector or adhesion molecules such as perforin, NKp44, granzyme, FasL, and TRAIL, and secrete IFN-γ to attach to and efflux tumor cells (Nair and Dhodapkar (2017). "Natural Killer T Cells in Cancer Immunotherapy." Frontiers in Immunology 8:1178). CIKs can enhance cytolytic activity toward tumor targets).

[0222] NKG2D is an activating receptor expressed on the surface of NK cells, CD8+ T cells, a subset of CD4+ T cells, type I NKT cells, and γδ T cells (see Lanier, LL, Cancer Immunol. Res. (2015) 3(6): 575-82). In humans and mice, NK cells express a heterodimer of CD94 and NKG2, two distinct type C lectin-like receptors that interact with non-polymorphic MHC class I analogs (HLA-E in humans and Qa1 in mice). HLA-E and Qa1 are unique in that, instead of binding to peptides derived from pathogens, they bind to fragments of signaling peptides derived from other MHC class I molecules during processing in the ER (Murphy, Kenneth M., et al. Janeways Immunobiology (See 9th ed., GS, Garland Science, Taylor & Francis Group, 2017, p. 129). This allows CD94:NKG2 to detect the presence of various different MHC class I variants whose expression can be targeted by viruses and to kill cells with reduced overall MHC molecule expression.

[0223] NKG2D plays a specific role in activating NK cells. Two NKG2D molecules form a homomer that binds to several MHC class I analogs induced by various types of cellular stress. These include the MIC molecules MIC-A and MIC-B, which are human-derived MHC class I-associated molecules expressed by stressed and malignant cells, and the RAET1 family of major histocompatibility complex (MHC) class I-associated genes; RAET1 functions as a ligand for the NKG2D receptor. The ligand for NKG2D is expressed in response to cellular or metabolic stress and is upregulated in early tumor cells that have transformed into malignancy, as well as in cells infected with intracellular bacteria and most viruses. Therefore, recognition by NKG2D acts as a generalized "danger" signal to the immune system. (Murphy, Kenneth M., et al.) Janeways Immunobiology . 9th ed., GS, Garland Science, Taylor& Francis Group, 2017. p. 130).

[0224] Natural death-like T cells (NKT)

[0225] NKTs are rapid responders of the immune system and mediate potent immunomodulatory and effector functions in various disease settings. Upon activation, NKTs can immediately initiate effector functions, such as cytokine secretion or cell lysis activity, without first differentiating into effector cells. This rapid response makes NKTs important players in the first line of innate defense. Additionally, many cytokines secreted by NKTs have a potent influence on αβ T cell differentiation and function, thereby linking NKTs to adaptive defense as well.

[0226] NKTs are cells that share morphological and functional characteristics with both conventional T cells and NKs. Although NKTs possess the T lineage and express the T-cell antigen receptor (TCR), which is characteristic of conventional T cells, they also express cell surface proteins characteristic of NKs. As such, they are considered a bridge between innate immunity and adaptive immunity.

[0227] NKTs can be found in almost every place where T cells and NKs are found, such as the spleen, liver, thymus, bone marrow, lymph nodes, umbilical cord blood, and peripheral blood. They typically make up less than 1% of the peripheral blood of human and non-human primates (Wah, MakTak, et al. “Chapter 11: NK, γδ T and NKT Cells.” Primer to the Immune Response. Elsevier, 2014). Activation of various types of NKTs results in various immunomodulatory responses, such as the ability to produce specific cytokines, the ability to activate other immune cells like normal T cells, NKCs, and / or DCs, and the ability to induce apoptosis through cytotoxic activity and / or expression, as well as the ability to interact with apoptosis-inducing effector molecules such as Fas ligands (FasL / Fas) and TNF-associated apoptosis-inducing ligands (TRAIL).

[0228] As shown in Table 3 below (Adapted from Godfrey et al. (2004). "NKT cells: what's in a name?" Immunology, Nature Reviews 4:231-237), three NKT cell subtypes (types I, II, and NKT-like cells) express different TCRs and TCR activations to facilitate various intracellular events that induce specific functional activities. Each classification contains many additional subtypes depending on its diverse phenotypes. Type 1 NKTs (also known as NKT-I, constant NKT, or iNKT) possess constant TCR α-chains with a restricted TCR β-chain repertoire. Type II NKTs (also known as diversity NKT, dNKT, variant NKT, vNKT, or NKT-II) are more diverse in that they express a wide range of different TCR chain combinations. Furthermore, studies suggest the existence of other NKT-like cells, such as CD1-restricted T-cells and MR1-restricted mucosal-associated constant T-cells (MAITs). Bennstein (2017), "Unraveling Natural Killer T-Cells Development" Front Immunol. 8:1950. However, most of what is known today concerns type I NKTs.

[0229] [Table 3]. Classification of Natural Killer T Cells

[0230]

[0231] *(m)-mouse; (h)- human

[0232] Similar to T cells, NKTs express diverse or semi-constant αβ TCRs, which are non-covalently coupled to the CD3 complex, a conserved multiple subunit signaling apparatus. However, unlike T cells expressing αβ TCRs that can be activated by peptide antigens bound to classical class I (CD8+) or class II (CD4+) MHC molecules, most NKTs respond to glycolipid antigens present on CD1d, a non-classical class I MHC-like molecule. (Seaman (2000) "Natural Killer Cells and Natural Killer T Cells." Arthirits & Rheumatism 43(6): 1204-1217). In other words, NKT TCRs recognize glycolipids, glycosphingolipids, or lipid structures presented on non-polymorphic CD1d molecules expressed by specialized and non-specialized APCs (antigen-presenting cells), including DCs, macrophages (Mo), B cells, thymus cells, adipocytes, hepatocytes, and endothelial cells. (Wah, MakTak, et al. "Chapter 11: NK, γδ T and NKT Cells." Primer to the Immune Response. Elsevier, 2014).

[0233] CD56 is present in some types of NKTs. As a form of neuronal adhesion molecule 1 (NCAM1), this molecule allows binding to other molecules. Therefore, molecules expressing CD56 can bind to each other via homoadhesion (meaning adhesion mediated by undefined adhesion molecules between the same cell types). Although CD56 is a typical phenotypic marker for natural death cells, it can actually be expressed in a wider range of immune cells, including αβ T cells, γδ T cells, DCs, and monocytes. (Van Acker, HH, et al., "CD56 in the immune system: more than a marker for cytotoxicity?" Front. Immunol. (2017) 8: 892).

[0234] Generally, NKT activation can be regulated by a balance of activating and inhibitory signaling. However, NK receptor expression by NKTs depends on the developmental stage of the NKT, its activation state, and the genetic background of the host (Wah, MakTak, et al. "Chapter 11: NK, γδ T and NKT Cells." Primer to the Immune Response. Elsevier, 2014). Activation can occur directly through the binding of appropriate antigens to the NKT TCR and CD1d molecules, or indirectly through induction via APCs.

[0235] Similar to NK cells, NKT stimulation and effector functions depend on the integration of signals derived from their various receptors. Through their cytotoxic functions, NKTs can recognize and kill virus-infected cells and neoplastic cells. NKTs can also initiate adaptive anti-tumor immunity by activating APCs. Additionally, NKTs can secrete anti-inflammatory cytokines that activate a series of NK and CTL killing actions.

[0236] NKTs can kill tumor cells through the perforin / granzyme pathway; the Fas / FasL pathway; and the TRAIL pathway. Activated NKTs can directly kill tumor cells by expressing perforin and Fas ligands on their surface. CD56+ NKTs appear to be more efficient apoptotic cells than CD56- ones. (Terabe, Masaki, and Jay A. Berzofsky. "Natural Killer T Cells Balancing the Regulation of Tumor Immunity." Springer New York, 2012, Ch. 5: The Regulation of CD1d+ and CD1d- Tumors by NKT Cells: The Roles of NKT Cells in Regulating CD1d+ and CD1d- Tumor Immunity" pp 71-93).

[0237] NKTs can also enhance ADCC mediated by NK cells. (Terabe, Masaki, and Jay A. Berzofsky. "Natural Killer T Cells Balancing the Regulation of Tumor Immunity." Springer New York, 2012, Ch. 5: The Regulation of CD1d+ and CD1d- Tumors by NKT Cells: The Roles of NKT Cells in Regulating CD1d+ and CD1d- Tumor Immunity" pp 71-93).

[0238] NKTs additionally perform an immunomodulatory role by stimulating the production of cytokines including interferon-γ (IFNγ), granulocyte giant colony-stimulating factor (GM-CSF), colony-stimulating factor 1 (CSF-1), tumor necrosis factor α (TNFα), transforming growth factor (TGFβ), interleukin (IL) 3 (IL-3), IL-5, IL-10, IL-13, chemokines (CCL1, 2, 3, 4) and (CXCL8) without limitation.

[0239] NKTs have been shown to regulate immune responses in many disease settings, including cancer, through direct cell lysis, immunomodulation of adaptive cell functions, and regulation of immunosuppressive cells. When activated, NKTs are stimulated in the spleen, liver, or bone marrow and undergo rapid clonal expansion within three days of antigen encounter. However, as discussed above, activated NKTs can immediately perform effector functions without the need for differentiation. Therefore, it has been noted that NKTs exist in a "pre-activated state" and provide timely and effective defense during the interval required by normal T cells for proliferation and differentiation into effectors of more finely customized adaptive responses.

[0240] NKTs play a role in the regulation of immunity through the release of cytokines. Since NKTs carry pre-formed mRNA for IL-4 and IFNγ, large quantities of these cytokines can be produced within 1–2 hours of activation. The secretion of IFNγ promotes a Th1 response, whereas the production of IL-4 promotes a Th2 response, particularly when cells come into contact with antibodies and CD3. Additionally, NKTs can synthesize IL-2, IL-10, and IL-17 among other interleukins, as well as TGFβ, TNFα, and a large array of chemokines.

[0241] Like NK cells, NKT cells express inhibitory and activating NK receptors, including NKG2D and CD94 / NKG2A in mice and humans, and specific KIR in humans. NKT cells also express CD40L, ICOS, and PD-1. (Wah, MakTak, et al. "Chapter 11: NK, γδ T and NKT Cells." Primer to the Immune Response. Elsevier, 2014).

[0242] Type I NKT

[0243] Type I NKT TCRs are primarily encoded by germline Va genes (Va14 / Ja18 in mice, Va24 / JaQ in humans) and additionally by more diverse non-germline VB chain genes (VB8.2 / 7 / 2 in mice, VB11 in humans). They respond to both α- and β-linked glycolipids and bind to CD1d in a parallel configuration containing primarily α-chains. (Kumar and Delovitch (2014) "Different subsets of natural killer T cells may vary in their roles in health and disease." Immunology 142: 321-336).

[0244] While the TCRα chain is essentially invariant across species and NKTs, the TCRβ chain can be diversified. For example, in humans, all NKTs express a TCR in which the TCRα chain is Va24 plus Ja18 and the TCRβ chain generally contains Vβ2, 7, or 8. Intracellular signaling is mediated by the associated CD3 complex. (Wah, MakTak, et al. "Chapter 11: NK, γδ T and NKT Cells." Primer to the Immune Response. Elsevier, 2014).

[0245] While CD1d-dependent activation is characteristic of NKT type I cells, NKT type I cells can also be stimulated in a CD1d-independent manner by exposure to various cytokines such as IL-12, IL-18, or IL-12 and type I IFN. (Kumar and Delovitch (2014) "Different subsets of natural killer T cells may vary in their roles in health and disease." Immunology 142: 321-336.)

[0246] Researchers were able to identify NKT type I cells in the laboratory based on their ability to recognize the exogenous antigen α-galactosylceramide (α-GalCer). In vivo, experimental administration of the antigen to mice activates these NKT cells, which then help promote tumor rejection or protect the animals against infection by various pathogens. (Wah, MakTak, et al. "Chapter 11: NK, γδ T and NKT Cells." Primer to the Immune Response. Elsevier, 2014).

[0247] Due to their ability to generate arrays of effector molecules, Type I NKTs inherently possess the potential to regulate the functions of all types of hematopoietic cells. Type I NKTs have been reported to regulate the functions of DCs, APCS such as macrophages and B cells, and other lymphocytes including NKs and other T cell subsets. (Liao et al. (2014) "The Functions of Type I and Type II Natural Killer I (NKT) Cells in Inflammatory Bowel Diseases." Inflamm Bowel Dis. 19(6): 1330-1338).

[0248] Type II NKT

[0249] Type II NKTs are more abundant in humans than Type I NKTs. Unlike Type I NKTs, Type II NKTs are inactive to α-GalCer or other α-linked glycolipids; instead, they are likely to recognize β-linked glycolipids such as sulfates, lysosulfates, lysophosphatidylcholine (Lyso-PC), and glucosylsphingosine (lyso-GL1). Most Type II dNKTs recognize naturally occurring autoantigens known as sulfates, which are abundant in various membranes, including myelin sheaths, in the central nervous system, pancreas, kidneys, and liver. Generally, Type II NKTs mediate protection from autoimmune diseases through the down-regulation of inflammatory responses induced by Type I NKTs. The TCRs of Type II NKTs are primarily encoded by non-germline type Va- and VB-chain genes. Type II NKTs typically come into contact with ligands possessing the B chain rather than the α chain, which is a mechanism of action shared by conventional T cells (convTCs), and to a lower degree with Type I NKTs. (Kumar and Delovitch (2014) "Different subsets of natural killer T cells may vary in their roles in health and disease." Immunology 142: 321-336).

[0250] Studies suggest that when stimulated with sulfatid, type II NKT has the ability to modulate the function of type I NKT.

[0251] NKT-like cells (e.g., cytokine-induced apoptosis (CIK) cells)

[0252] NKTs expressing a combination of CD3 (T cell marker) and CD56 (NK marker) are a major cytotoxic subset of NKTs that may also be known as cytokine-induced apoptosis (CIK) cells. The phenotype of CIK cells is CD3 + CD56 + , CD3 + CD56 - and CD3 -CD56 + Although varying among them, they may not express the Fc receptor CD16. Like other series of apoptotic cells, NKT-like cells possess immunomodulatory effector functions. (Gutegemann et al. (2007). "Cytokine-induced killer cells are type II natural killer T cells." GMS German Medical Science 5: 1-4).

[0253] CIK and NKT-like cell differentiation from type I and type II NKTs is not well understood.

[0254] Cytokine-induced apoptosis (CIK)

[0255] CIK cells are a heterogeneous population of CD8+ T cells capable of expanding in vitro through incubation with anti-CD3 antibodies, IFN-γ, and IL-2. They possess cytotoxic activity mediated by FasL / Fas and perforin / granzyme action. CIKs are generally divided into two major subsets: CD3+CD56+ types and CD3+CD56- types. CD3+CD56+ T cells (NKT cells) are considered the primary effector cells of CIKs. CIK cells can elute cancer cells in an MHC-unrestricted manner by activating NK cell receptors such as DNAX accessory molecule-1, NKp46, NKG2D, and NKp30. Several studies have shown that levels of anti-inflammatory cytokines secreted by CIK cells, such as tumor necrosis factor (TNF)-α, IFN-γ, and IL-2, are significantly upregulated following stimulation by tumor cells; These cytokines were shown to further enhance systemic antitumor activity and induce a Th1 immune response. (Gao, et al. "Cytokine-Induced Killer Cells As Pharmacological Tools for Cancer Immunotherapy." Frontiers, Frontiers, 19 June 2017, www.frontiersin.org / articles / 10.3389 / fimmu.2017.00774 / full).

[0256] γδ T cells (GDT)

[0257] γδ T cells (GDTs) are another population of apoptotic cells that bridges the gap between innate and adaptive immunity. Despite belonging to the T cell lineage and expressing surface TCRs, γδ T cells also display the NK receptor NKG2D and exhibit non-specific recognition that activates its cytotoxic and immunomodulatory effector functions. (Wu YL, Ding YP, Tanaka Y, Shen LW, Wei CH, Minato N, Zhang W. γδ T Cells and Their Potential for Immunotherapy. Int J Biol Sci 2014; 10(2):119-135. doi:10.7150 / ijbs.7823. Available from http: / / www.ijbs.com / v10p0119.htm).

[0258] GDTs can be organized into two major populations distinguished by the surface expression of αβ TCRs (discussed below) and γδ TCRs. T cells expressing αβ TCRs generally also express CD4 or CD8 lineage markers (discussed above). However, GDTs generally do not express these markers and do not require conventional antigen presentation in the context of MHC presentation. GDTs can be further organized into populations based on δ chain expression, specifically Vδ1, Vδ2, and Vδ3 chains. Similar to the combinatorial diversity of the αβ TCR repertoire, the diversity of GDTs is at least as great as that of the αβ TCR repertoire due to extensive non-genetic mechanisms of action. Id.

[0259] Similar to other series of apoptotic cells, GDTs function as lysis / cytotoxic effectors through the perforin / granulicin-granzyme pathway as well as the apoptotic receptor / ligand pathway, such as Fas / FasL. Furthermore, GDTs secrete Th1, Th2, and Th17 cytokines, each of which exerts immunomodulatory effects on innate and adaptive immunity.

[0260] Cellulose T lymphocytes (CTL)

[0261] Cellulosic T lymphocytes (CTLs) constitute a distinct lymphocyte subpopulation. Unlike NKTs, they are induced by a variety of stimuli, including major histocompatibility antigens, protein antigens, viruses, and intracellular bacteria and peptides.

[0262] Like many other cells discussed in this specification, CTLs have cell lysis effector functions. However, unlike NK and NKTs, CTLs release cytotoxic proteins perforin and granzyme in an antigen-dependent manner.

[0263] CD8+ CTL

[0264] Unlike some NKTs and NKs, CD8+ CTLs recognize peptides bound to class I MHC. The activation and proliferation of CTLs are induced by exposure to specific antigens. Similar to NKTs, the activation of CD8+ CTLs leads to the secretion of cytolytic mediators, such as perforins and granzymes, which induce apoptosis in target cells, such as tumor cells. This action is cell-specific: studies suggest that CD8+ CTLs redirect their secretory apparatus toward each cell and attack only one contact point at a time. (Murphy, Kenneth M., et al.) Janeways Immunobiology . 9th ed., GS, Garland Science, Taylor& Francis Group, 2017. pps. 387-395).

[0265] Due to their additional similarities to NKTs, CTLs secrete various cytokines, such as IFNy and TNF-α, which enhance antigen presentation and mediate antipathogenic effects. IFN-α induces increased expression of MHC class I and other molecules involved in the peptide incorporation of newly synthesized MHC class I proteins in infected cells. This increases the likelihood that target cells will be recognized for cytotoxic attacks. TNF-α acts synergistically with IFN-α to enhance its effector function. Various cytokines, such as IFNy, which generate IL-2 or CD4+ T cells, have been reported to be necessary for the generation of effective CTL immunity (Ito and Seishima (2010), "Regulation of Induction and Function of Cytotoxic T Lymphocytes by Natural Killer T Cell." J Biomed Biotechnol, Art. ID. 641757).

[0266] The induction of CD8+ CTLs and the enhancement of activation through type I NKT activation lead to immune enhancement against tumors or microorganisms. As discussed above, activation of type I NKTs by IL-12 (CIK) or α-GalCer (non-CIK NKTs) can result in Th1 cytokine production (IFNy). However, studies indicate that activation of type I NKTs by α-GalCer can result in NKTs that secrete both Th1 cytokines (IFNy) and Th2 cytokines (IL-4). On the other hand, activated type II NKTs inhibit CD8+ CTL activation through cytokine production, namely IL-4, IL-13, and TGF-B. (Ito and Seishima (2010), "Regulation of Induction and Function of Cytotoxic T Lymphocytes by Natural Killer T Cell." J Biomed Biotechnol, Art. ID. 641757).

[0267] CD8+ CTLs are activated when naive CD8+ T cells interact with APCs. This activation depends on the number of MHC complexes present on the APC, the affinity of the CTL's TCR for the MHC complexes, and the signals provided by the APC in the form of co-stimulatory molecules. The B7 / CD28 and CD40 / CD40L co-stimulatory pathways are well-known examples of co-stimulatory interaction pathways that contribute to T cell activation and production. NKT activation can upregulate co-stimulatory molecules (e.g., CD40, CD80, and CD86) in both CD8+ and CD8- DC subsets. Both the B7 / CD28 and CD40 / CD40L co-stimulatory pathways are additionally linked to NKT activation. (Ito and Seishima (2010), "Regulation of Induction and Function of Cytotoxic T Lymphocytes by Natural Killer T Cell." J Biomed Biotechnol, Art. ID. 641757).

[0268] Therefore, various stimulants of NKT indicate the production of different types of cytokines that regulate the level of CTL activation.

[0269] CD4+ CTL

[0270] Little is known about the phenotype, function, and transcriptional profile of cytolytic CD4+ T cells. Similar to NKTs, CD4+ CTLs exhibit numerous markers and effector functions across adaptive and innate immunity.

[0271] Despite being phenotypically Th1 lineage cells, the cell marker profile of CD4+ T cells differs from that of other Th1 lineage cells. For example, CD4+ cells lose the ability to secrete IL-2, lack the expression of CD28 and CD27, and further upregulate the expression of CD57 as well as the integrin α chains CD11a and CD11b. Similar to NK cells, cytolytic CD4+ cells express NKG2D, KIR2DS2, and KARAP / DAP12 receptors; NKG2D has been hypothesized to act as a receptor in cytolytic CD4+ T cells lacking CD28. Additionally, CTLs express CD25, a cell marker characteristic of Tregs. (Soghoian, Damien Z, and Hendrik Streeck. “Cytolytic CD4( ) T Cells in Viral Immunity.” Expert Review of Vaccines, US National Library of Medicine, Dec. 2010, www.ncbi.nlm.nih.gov / pmc / articles / PMC3033049 / ).

[0272] Cell-lysing CD4+ cells were shown to additionally express FasL and induce apoptosis in targets expressing Fas. CD4+ CTLs were shown to additionally express higher levels of perforin and granzyme or granulicin and elute target cells in an antigen-dependent manner. The degranulation of these cells by antigen stimulation may be enhanced by the availability of IL-2 potentially secreted by a different series of apoptotic cells. Additionally, the study indicated that CD4+ CTLs expressing TRAIL can induce bystander apoptosis in antigen-presenting cells as well as TRAIL-sensitive tumor cell lines. Id.

[0273] CD4+ CTLs also perform an immunomodulatory role as they have been found to secrete TNF-α and INF-γ. As discussed above, target cells can be sensitized to TRAIL-mediated apoptosis by the presence of anti-inflammatory cytokines. These cytokines can enhance apoptosis indirectly through direct, contact-independent cytotoxic mechanisms, either by activating cytolytic activity by inducing the production of NO and other free radicals or by activating apoptotic pathways within tumor cells. Id.

[0274] Mucosa-associated constant T cells (MAIT cells)

[0275] MAIT cells are invariant Vα7.2 TCR-expressing cells that exhibit an immune response against tumor cells similar to NKTs. Unlike NKTs, MAIT cells are stimulated by MR1, a non-classical MHC class Ib molecule encoded by chromosome 1 similar to CD1d. Several lines of evidence suggest that MR1 presents ligands to MAIT cells, possibly glycolipids, similar to how it stimulates NKT cells. Furthermore, it is also activated in a co-receptor-independent mode. MAIT cells possess both peripheral CD56+ and CD56- subsets, but the CD56- subset is thought to be linked to tumor-infiltrating T cells. The CD56- subset of MAIT cells is unrelated to IL-4, IL-5, and IL-10 but correlates with the expression of anti-inflammatory cytokines. (Peterfalvi, et al. “Invariant Vα7.2-Jα33 TCR Is Expressed in Human Kidney and Brain Tumors Indicating Infiltration by Mucosal-Associated Invariant T (MAIT) Cells.” OUP Academic , Oxford University Press, 16 Oct. 2008, academic.oup.com / intimm / article / 20 / 12 / 1517 / 684337).

[0276] Lympokin-activated apoptotic cells (LAK)

[0277] Lymphokine-activated apoptotic cells (LAKs) are cytotoxic effector cells with an exceptionally broad spectrum of target cells, including normal and malignant cells of different origins. They are apoptotic lymphocytes activated in the presence of interleukin-2 (IL-2). LAK cells exhibit profound heterogeneity with respect to phenotypic surface marker expression; it has not yet been determined whether they represent a distinct cell lineage.

[0278] Activation of a series of dead cell populations

[0279] A series of apoptotic cells can directly or indirectly recognize histocompatibility molecules between genetically disparate individuals within the same species, also known as "homologous recognition." Some series of apoptotic cells can recognize determinants for MHC, MHC-like complexes, and other molecules displayed on the surface of host cells (i.e., tumor-infiltrated cells) via direct homologous recognition without the need for antigen processing. Some series of apoptotic cells can recognize processed peptides of antigens presented by MHC molecules in a self-limiting manner via indirect homologous recognition. While innate populations of apoptotic cells may be activated by a single mechanism of action of homologous recognition (e.g., direct homologous recognition), some series of apoptotic cells require multiple stimulus signals to induce effector functions (i.e., both indirect and direct homologous recognition). Additionally, there are numerous inhibitory receptors present on the surface of apoptotic cells that suppress apoptotic cell functions. Therefore, the activation of a series of apoptotic cell effector functions may additionally require the prevention of inhibitory receptor ligand binding on the surface of host cells or in the response environment, interference with inhibitory receptors on the surface of apoptotic cells, or the blocking of inhibitory signals induced in apoptotic cells. (Benichou, G, and AW Thomson. "Direct versus Indirect Allorecognition Pathways: on the Right Track.") American Journal of Transplantation: Official Journal of the American Society of Transplantation and the American Society of Transplant Surgeons , US National Library of Medicine, Apr. 2009, www.ncbi.nlm.nih.gov / pmc / articles / PMC3746751 / ).

[0280] The majority of T cells recognize linear peptides presented by cell-surface MHC class I or class II molecules and / or MHC-like molecules. However, most killer T cells must first be stimulated by specialized APCs and subsequently activated by receiving signals from co-stimulatory molecules and cytokines.

[0281] Natural cytotoxic receptors (NCRs) are present on the surface of a series of apoptotic cells, such as NK cells, NKT cells, GDT cells, macrophages, CD4+ CTLs, and CD8+ CTLs. One example, NKG2D, is a C-type lectin-like type II transmembrane glycoprotein. Other natural cytotoxic receptors include, without limitation, NKp46 (NCR1, CD335), NKp44 (NCR2, CD336), and NKp30 (NCR3, CD337). These natural cytotoxic receptors act as activators of a series of apoptotic cells or as co-stimulatory signals in heterogeneous cross-reactive activation.

[0282] Exogenous microbial components known as pathogen-associated molecular patterns (PAMPs), or endogenous inflammatory factors released from necrotic cells known as damage-associated molecular patterns (DAMPs), bind to germline-encoding pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), nucleotide oligomerization domain (NOD)-like receptors (NLRs), and C-type lectin receptors (CLRs). Immature dendritic cells recognize various arrays of PAMPs via TLRs present on their cell surface. Upon detecting the presence of PAMPs, immature DCs transform into mature DCs, resulting in increased surface levels of MHC proteins and co-stimulatory molecules. The efficacy of this maturation process aids in priming naive T cells and also helps primate NK cells. (Ebihara, et al., "Induction of NKG2D Ligands on Human Dendritic Cells by TLR Ligand Stimulation and RNA Virus Infection.") OUP Academic , Oxford University Press, 18 Sept. 2007, academic.oup.com / intimm / article / 19 / 10 / 1145 / 743680).

[0283] Triggering of PAMPs by TLRs on APCs results in the production of Type I IFN, IL-12, IL-18, and IL-15, which help prime a series of apoptotic cells for activation.

[0284] Triggering of APCs by PRRs results in multiple immune system effects: 1) high expression of stable "non-auto" proteins presenting MHC complexes on cell surfaces that trigger T cell responses; 2) high expression of co-stimulatory molecules such as CD80 and CD86 that prime and activate antigen-specific T cells; and 3) secretion of anti-inflammatory cytokines such as IL-1, IL-6, IL-12, TNF-α, GM-CSF, and IFN-γ. The secretion of anti-inflammatory cytokines then induces the activation of APCs, which helps directly activate a non-specific series of apoptotic cells and promote T-cell differentiation into antigen-specific helper T cells or cytotoxic T cells that mediate acquired immunity. (Mendelsohn, John, et al. The Molecular Basis of Cancer. Elsevier Health Sciences, 2015, pp. 695-739).

[0285] The same TLRs that identify PAMPs can also be activated by DAMPs. DAMPs are molecules not derived from pathogens, but instead are molecules similar to the intracellular contents of cells that have undergone apoptosis. There is evidence that DAMP signaling via TLRs initiates and amplifies pathogen-independent responses. In the context of normal tissue surveillance, NKG2D ligand expression indicates cellular stress and displays self-identification marks for cytotoxic lymphocytes. NKG2D ligand expression has been found to be upregulated by stimulation of immature DCs with PAMPs. Similarly, DAMPs also indicate "danger" signals; in the context of cell damage, a correlation has been shown between TLR activation and increased expression of NKG2D ligands. It has been suggested that NKG2D activation occurs in response to cell damage, causing innate cells such as NK cells to become hypersensitive to future activation. (Wortham, Brian W., et al., "TLR and NKG2D Signaling Pathways Mediate CS-Induced Pulmonary Pathologies.") PLOS ONE , Public Library of Science, journals.plos.org / plosone / article?id=10.1371 / journal.pone.0078735).

[0286] Ultimately, the binding of PAMPS to PRRs triggers the activation of the MAPK cascade through adapter proteins, myeloid differentiation primary-response protein 88 (MyD88), and T1R domain-containing adapter proteins that induce IFNβ (TRIF (Qian, F. et al., (2016) "Pivotal role of mitogen-activated protein kinase-activated protein kinase 2 in inflammatory pulmonary diseases," Curr. Protein Pept. Sci. 17(4): 332-42; citing Qian, C. and Cao, X, (2013), "Regulation of Toll-like receptor signaling pathways in innate immune responses," Ann. NY Acad. Sci. 1283: 67-74).

[0287] In normative signaling, p38 MAPK is selectively phosphorylated by MAPKKs (MKK3 and MKK6), which in turn are activated by MAPKKKs including TGFβ-activated kinase 1 (TAK1), apoptosis signal-regulating kinase 1 (ASK1), mixed-lineage kinase 2 (MLK2), or MLK3. p38 MAPK-mediated signaling initiates the activation of several transcription factors, including CREB, ATF2, and Myc, as well as other kinases, including MK2, MK3, MNK1 / 2, and MSK1 / 2 (cited in Obata, T. et al., (2000) Crit. Care Med. 28 (4 Suppl.: N67-N77; Dong, C. et al., (2002) "MAP kinases in the immune response," Annu. Rev. Immunol. 20: 55-72). Among these terminal kinases, the role of MK2 has been determined to be essential for the regulation of innate immune responses, including the regulation of the production of inflammatory cytokines and chemokines, reactive oxygen species (ROS), and nitric oxide (NO).

[0288] As used herein, the term "stable" refers to resistance to molecular or chemical changes. In the context of the ENLST™ cells of the described invention, it refers to a population of stably transfected ENLST™ cells that generate reproducible levels of MNC immunostimulation in a dose-dependent manner in in vitro MLTRs. It comprises stably transfected living ENLST™ cells, membrane fragments of ENLST™ cells containing exogenous immunomodulatory proteins, and dead necrotic ENLST™ cells containing immunomodulatory proteins expressed on their surfaces, each capable of directly or indirectly stimulating MNCs.

[0289] The term “stem cell” refers to an undifferentiated cell having high proliferative potential and the ability to self-renewal to generate daughter cells capable of undergoing terminal differentiation into more than one distinct cell phenotype. In this specification, the terms “renewal” or “self-renewal” refer to the process by which a stem cell divides to generate one (asymmetric division) or two (symmetric division) daughter cells having developmental potential indistinguishable from the parent cell. Self-renewal includes both proliferation and maintenance of the undifferentiated state.

[0290] As used herein, the term "stimulate" and any various grammatical forms refer to inducing activation or increasing activity.

[0291] The terms “object,” “entity,” or “patient” as used in this specification are used interchangeably to refer to members of animal species of mammalian origin, including humans.

[0292] As used herein, the term “subjects requiring this” refers to a patient who, unless the context and usage of the phrase otherwise indicate, (i) is to be administered the composition according to the described invention, (ii) is receiving the composition according to the described invention, or (iii) has received the composition according to the described invention. “Subjects requiring this” may also refer to subjects who have or are suspected of having a disorder sensitive to treatment using the composition of the described invention.

[0293] With respect to the cellular components of the composition as used herein, the term “substantially pure” refers to the cellular components being substantially separated from any material that may be associated with them in a living system. This refers to a purity of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% as determined by an analysis protocol. Such protocols may include, but are not limited to, FACS, for example.

[0294] In any grammatical form used herein, the term "inhibition" refers to inhibiting or reducing activity.

[0295] As used herein, the term “symptom” refers to a sign or indication of a disorder or disease, particularly when experienced by an individual as a change from normal function, sensation, or appearance.

[0296] As used herein, the term “therapeutic agent” refers to a drug, molecule, nucleic acid, protein, metabolite, cell, composition, or other substance that provides a therapeutic effect. As used herein, the term “active” refers to an element, component, or constituent of the composition of the described invention responsible for the intended therapeutic effect. The terms “therapeutic agent” and “active agent” are used interchangeably herein. As used herein, the term “therapeutic component” refers to a therapeutically effective dose (i.e., dose and frequency of administration) that eliminates, reduces, or prevents the progression of a specific disease sign in a percentage of the population. An example of a commonly used therapeutic component is ED50, which describes a dose at a specific dosage that is therapeutically effective for a specific disease sign in 50% of the population.

[0297] The terms "therapeutic dose," "effective dose," or "pharmaceutical effective dose" of an active agent are used interchangeably to refer to an amount sufficient to provide the intended benefit of treatment. However, dosage levels are based on various factors, including age, weight, gender, the patient's medical status, the severity of the condition, the route of administration, and the specific active agent used. Therefore, while dosage regimens can vary significantly, they can be routinely determined by a physician using standard methods. Additionally, the terms “therapeutic dose,” “and pharmaceutically effective dose” include a prophylactic or preventive amount of the composition of the described invention. In the prophylactic or preventive application of the described invention, the pharmaceutical composition or agent is administered to a patient who is susceptible to or otherwise at risk of a disease, disorder, or condition in an amount sufficient to eliminate or reduce the risk, reduce the severity, or delay the onset of the disease, disorder, or condition, including biochemical, histological, and / or behavioral symptoms of the disease, disorder, or condition or its complications, and intermediate pathological phenotypes that appear during the development of the disease, disorder, or condition. Generally, it is desirable to use the maximum dose according to some medical judgment, i.e., the safest maximum dose. The terms “dose” and “administration amount” are used interchangeably herein.

[0298] As used herein, the term "therapeutic effect" refers to the results of treatment, which are deemed desirable and beneficial. Therapeutic effects may include, directly or indirectly, the cessation, reduction, or elimination of signs of disease. Therapeutic effects may also include, directly or indirectly, the cessation, reduction, or elimination of the progression of signs of disease.

[0299] For any therapeutic agent described herein, the therapeutic effective dose may be determined initially from preliminary in vitro studies and / or animal models. The therapeutic effective dose may also be determined from human data. The applicable dose may be adjusted based on the relative bioavailability and efficacy of the administered compound. Adjusting the dose to achieve maximum efficacy based on the methods described above and other well-known methods is typically within the scope of ability of a skilled technician.

[0300] The general principles for determining therapeutic effects found in Chapter 1 of Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th Edition, McGraw-Hill (New York) (2001), incorporated herein by reference, are summarized below.

[0301] Pharmacokinetic principles provide a basis for modifying dosage regimens to achieve the desired level of therapeutic efficacy while minimizing unacceptable side effects. In situations where the plasma concentration of a drug can be measured and related to the therapeutic window, additional guidance for dosage adjustment can be obtained.

[0302] Drug products are considered pharmaceutical equivalents if they contain the same active ingredient and have the same strength or concentration, dosage form, and route of administration. Two pharmaceutically equivalent drug products are considered biological equivalents if the ratio and degree of bioavailability of the active ingredient in the two products do not differ significantly under appropriate test conditions.

[0303] The term "therapeutic window" refers to the concentration range that provides therapeutic efficacy without unacceptable toxicity. Following the administration of a specific dose of a drug, the effect generally exhibits a characteristic transient pattern. There is a delay period before the drug concentration exceeds the minimum effective concentration ("MEC") for the desired effect. Following the onset of the response, the intensity of the effect increases as the drug continues to be absorbed and distributed. This results in a decrease in the intensity of the effect as it reaches a peak and then disappears when the drug concentration drops back below the MEC due to drug elimination. Therefore, the duration of the drug's action is determined by the period during which the concentration exceeds the MEC. The therapeutic goal is to obtain and maintain the concentration within the therapeutic window for the desired response with minimal toxicity. A drug response below the MEC for the desired effect will be less therapeutic, whereas the potential for toxicity in the case of side effects will increase above the MEC. Increasing or decreasing the drug dosage shifts the response curve up or down the intensity scale and is used to regulate the drug's effect. Increasing the dose also extends the duration of the drug's action but carries the risk of an increased potential for side effects. Therefore, unless the drug is non-toxic, increasing the dosage is not a useful strategy for extending the duration of action.

[0304] Instead, different doses of the drug must be administered to maintain concentrations within the therapeutic window. Generally, the lower limit of a drug's therapeutic range appears to be approximately equal to the drug concentration that produces about half of the maximum possible therapeutic effect, and the upper limit is set such that about 5% to 10% or fewer of patients will experience toxic effects. These figures can be highly variable; some patients may benefit greatly from drug concentrations exceeding the therapeutic range, while others may experience significant toxicity at much lower values. The therapeutic goal is to maintain steady-state drug levels within the therapeutic window. For most drugs, the actual concentration associated with this desired range is unknown and does not need to be known; it is sufficient to understand that efficacy and toxicity are generally concentration-dependent and how drug dosage and frequency of administration affect drug levels. For a small number of drugs where there is a small (2- to 3-fold) difference between the concentrations causing efficacy and toxicity, a plasma concentration range associated with effective treatment has been defined.

[0305] In this case, a target level strategy is reasonable, where a desired target steady-state concentration of the drug (typically in plasma) associated with efficacy and minimal toxicity is selected, and the dosage expected to achieve this value is calculated. The drug concentration is subsequently measured, and if necessary, the dosage is adjusted to be closer to the target.

[0306] In most clinical situations, drugs are administered as a series of repeated doses or continuous infusions to maintain steady-state concentrations associated with a therapeutic window. To maintain a selected steady-state or target concentration ("maintenance dose"), the rate of drug administration is adjusted so that the infusion rate is equal to the rate of loss. If a clinician selects the desired plasma drug concentration and knows the clearance and bioavailability of the drug in a specific patient, they can calculate the appropriate dose and dosing interval. However, living cell therapies break this concept because, in the case of autologous cell therapy, the cells are divided and, moreover, can maintain a permanent presence in the body. Therefore, what is initially administered may have little correlation with what remains in the receptor over time.

[0307] As used herein, the term “treating” includes abolishing, substantially inhibiting, slowing, or reversing the progression of a condition, substantially improving the clinical symptoms of the condition, or substantially preventing the onset of clinical symptoms of the condition. Treating further refers to achieving one or more of the following: (a) reducing the severity of the disorder; (b) limiting the development of characteristic symptoms of the disorder(s) being treated; (c) limiting the exacerbation of characteristic symptoms of the disorder(s) being treated; (d) limiting the recurrence of the disorder(s) in patients who previously had the disorder(s); and (e) limiting the recurrence of symptoms in patients who were previously asymptomatic for the disorder(s). Treating also includes eliciting a clinically significant response, whether detectable or not, without excessive levels of adverse effects.

[0308] The terms "tumor load" and "tumor load" are used interchangeably to refer to the number of cancer cells, the size of a tumor, or the amount of cancer in the body.

[0309] As used herein, the terms “wild type,” “naturally occurring,” or their grammatical equivalents mean an amino acid sequence or nucleotide sequence found in nature and containing allelic variations; that is, an amino acid sequence or nucleotide sequence that is generally not intentionally modified. Accordingly, the terms “non-naturally occurring,” “synthetic,” “recombinant,” or their grammatical equivalents are used interchangeably to refer to an amino acid sequence or nucleotide sequence not found in nature; that is, an amino acid sequence or nucleotide sequence that is generally intentionally modified. It is understood that once recombinant nucleic acid is created and reintroduced into a host cell or organism, it will be replicated non-recombinantly, that is, using the in vivo cellular machinery of the host cell rather than in vitro manipulation; however, once produced by recombination, such nucleic acid is still considered recombinant for the purposes of the described invention, even if it is subsequently replicated non-recombinantly.

[0310] Method for preparing anti-tumor cell therapy for passive immunization of cancer patients

[0311] According to one embodiment, the described invention provides a method for passive immunization of a cancer subject not currently under the influence of immunosuppressive therapy, comprising a cell product containing activated and expanded mononuclear cells comprising a series of apoptotic cells and an expanded subpopulation following in vitro activation of a cytotoxic T-cell population, said method comprising, under sterile conditions, the following steps:

[0312] Step 1 : Steps for inducing an in vitro immune response by the following steps:

[0313] (a) A step of isolating a population of mononuclear cells (MNCs) from a biological sample;

[0314] (b) a step of preparing a population of engineered leukocyte-stimulating cells ("ENLST™ cells") comprising allogeneic tumor cell lines transfected or transduced with a recombinant DNA sequence encoding at least three essential immunomodulatory peptides, wherein the three essential immunomodulatory peptides are an OX40 ligand (OX40L), a CD27 ligand (CD70), and a CD28 ligand (CD28L) comprising CD80, CD86, or both;

[0315] (c) a step of inducing an immune response by contacting the population of MNCs of step (a) with the ENLST™ cells of step (b) in vitro, comprising a population of activated MNCs containing a series of activated apoptotic cells effective in killing tumor cells;

[0316] Step 2 : A step of expanding an activated population of MNCs containing an activated subpopulation of a series of apoptotic cells in vitro by culturing the activated MNCs to form a cell product;

[0317] Step 3 : A step of preparing a unit dose package containing individual doses of cell products; a step of freezing the unit package containing the cell products at -86°C and freezing the frozen unit dose package in the vapor phase of a liquid nitrogen freezer (hereinafter "freezing storage");

[0318] Step 4 : A step of thawing a therapeutic dose of a frozen unit dose package containing a cell product under controlled conditions; and optionally, a step of combining the frozen and thawed cell product of step 4 with a pharmaceutically acceptable carrier component to form a pharmaceutical composition; and

[0319] Step 5 : A step of administering a therapeutic amount of the cell product of step 4 or the pharmaceutical composition containing the activated and expanded cell product to a subject.

[0320] According to some embodiments, the immune system of a cancer subject not currently under the influence of immunosuppressive therapy is intact, which means that the immune system is not depleted by immunosuppressive therapy. According to some embodiments, immunosuppressive therapy includes chemotherapy. According to some embodiments, the subject is a melanoma patient. According to some embodiments, the subject is a prostate cancer patient. According to some embodiments, the subject is a breast cancer patient.

[0321] Step 1: Step to induce an in vitro immune response

[0322] (i) Step of isolating a population of mononuclear cells (MNCs) from a biological sample

[0323] According to some embodiments, the mononuclear cells are derived from a biological sample. According to some embodiments, the biological sample is autologous tissue to the recipient subject. According to some embodiments, the biological sample is homologous to the recipient subject. According to some embodiments, the biological sample is derived from a subject who is not currently a recipient and is not under the influence of immunosuppressive therapy. According to some embodiments, the biological sample is of mammalian origin. According to some embodiments, the biological sample is human. According to some embodiments, the source of the mononuclear cell population is body fluid. According to some embodiments, the body fluid is umbilical cord blood, whole blood, peripheral blood, mobilized peripheral blood, or bone marrow. According to some embodiments, the source of the mononuclear cell population is whole blood. According to some embodiments, the biological sample is a bone marrow sample. According to some embodiments, the biological sample is umbilical cord blood.

[0324] According to some embodiments, the biological sample is a peripheral blood sample. According to some embodiments, the sample is a mobilized peripheral blood sample. Treatment with hematopoietic growth factors has been shown to cause a significant increase in the number of hematopoietic progenitor cells in peripheral blood, when measured by the presence of CD34+ cells or in colony formation assays in CFU. These mobilized peripheral blood hematopoietic stem cells (HSCs) are used in transplantation, immunotherapy, and cardiovascular regenerative medicine. For example, colony-stimulating factors are agents used for the mobilization of hematopoietic stem cells. Examples of colony-stimulating factors include, without limitation, G-CSF, GM-CSF, and pharmaceutically acceptable analogs and derivatives thereof. For example, filgrastim, a G-CSF analog produced by recombinant technology, is sold under the brand names Neupogen® (Amgen); Religrast® (Reliance Life Sciences); Nugraf® (Zenotech Laboratories, Ltd.); and Neukine® (Intas Biopharmaceuticals).

[0325] According to some embodiments, monocytes may be isolated from whole blood by density gradient centrifugation using a hydrophilic colloid (e.g., a polymer formed by the copolymerization of sucrose and epichlorohydrin (Ficoll-Paque®) or polyvinylpyrrolidone-coated colloidal silica (Percoll®). In an exemplary protocol, a diluted mixture of PBS and peripheral blood is layered in a top-mounted 50 ml centrifuge tube of Ficoll-Paque® and centrifuged at 400 xg for 30–40 minutes at 20°C on a brakeless swing-bucket rotor. The supernatant is aspirated so that the monocyte layer (lymphocytes, monocytes, and platelets) at the interface is undisturbed. The monocyte layer is carefully transferred to a new 50 ml centrifuge tube. The cells are washed with PBS (pH 7.2) containing 2 mM EDTA and centrifuged at 300 for 10 minutes at room temperature. Centrifuge at xg and discard the supernatant. To remove platelets, resuspend the cell pellet in 50 mL of buffer and centrifuge at 200 xg for 10-15 minutes at room temperature. Remove the supernatant containing platelets. This step is repeated. The cell pellet is resuspended in a suitable buffer solution or medium for downstream application.

[0326] An alternative exemplary protocol for isolating PBMCs is through leukocyte apheresis. For example, whole blood can be obtained from a patient with informed consent and processed using a device that automatically separates the target PBMC fraction from other components of the blood, such as plasma and red blood cells; while the isolated PBMCs are collected, the other components are returned to the patient. The collected PBMCs may undergo further processing, for example, the removal of residual red blood cells through elution.

[0327] According to some embodiments, mononuclear cells may also be isolated from an allogeneic source, e.g., cord blood. The mononuclear cell (MNC) fraction of cord blood consists of lymphocytes (T cells, B cells, and NK cells), monocytes, dendritic cells, and stem / progenitor cells. Anticoagulant cord blood citrate phosphate dextrose (Sigma-Aldrich, St. Louis, Missouri) is drawn directly into a 50 mL tube containing 5 mL of buffer and stored at 4°C prior to isolation. The anticoagulant cord blood is diluted to three times the volume of the buffer. The diluted cell suspension is carefully stratified onto a Ficoll-Paque® in a 50 mL conical tube and centrifuged at 400 xg for 35 minutes at 20°C in a brake-free swing bucket rotor. The supernatant is aspirated so that the mononuclear cell layer remains undisturbed during interphase. The mononuclear layer is carefully transferred to a new 50 mL conical tube. Fill the tube with buffer, mix, and centrifuge at 300 xg for 10 minutes at 20°C. Carefully aspirate the supernatant. To remove platelets, resuspend the cell pellet in 50 mL of buffer and centrifuge at 200 xg for 10–15 minutes at 20°C. Carefully and completely remove the supernatant. The cell pellet is resuspended in an appropriate amount of buffer for downstream application.

[0328] According to some embodiments, mononuclear cells may be isolated from bone marrow: Bone marrow is collected from the superior iliac crest or sternum using an aspiration needle. The aspirated human bone marrow is diluted with an appropriate buffer at a ratio of 7:1. The cells are passed through a 100 μm filter to remove bone fractions and cell clumps. The diluted cell suspension is layered on Ficoll-Paque® in a 50 ml conical tube and centrifuged at 445 xg for 35 minutes at 20°C in a brakeless swing bucket rotor. The supernatant is aspirated so that the mononuclear cell layer remains undisturbed. During the interphase, the bone marrow MNCs are carefully transferred to a new 50 ml conical tube. The cells are washed with buffer, gently mixed, and centrifuged at 300 xg for 10 minutes at 20°C. The supernatant is carefully removed. To remove platelets, the cell pellet is resuspended in 50 ml of buffer and centrifuged at 200 xg for 10-15 minutes at 20°C. Carefully remove the supernatant. The cell pellet is resuspended in a suitable buffer for downstream application.

[0329] According to some embodiments, the isolated population of MNCs comprises a mixed population of lymphocytes, a population of monocytes, and a population of dendritic cells. In humans, the frequency of these populations in peripheral blood varies by individual, but typically lymphocytes are in the range of 70-90%, monocytes are in the range of 10-20%, while dendritic cells are rare, accounting for only 1-2%. (Kleiveland, CR, "Peripheral Blood Monouclear Cells" in: Verhoeckx, K. et al. (eds). The Impact of Food Bioactives on Health (2015), Springer, Cham. Doi.org / 10.1007 / 978-3-319-1610404_15) According to some embodiments, the mixed population of lymphocytes comprises a subpopulation of T cells, a subpopulation of B cells, and a subpopulation of NK cells. According to some embodiments, T lymphocytes include a subgroup of CD8 T lymphocytes and a subgroup of CD4 T lymphocytes.

[0330] (ii) Preparation of a population of engineered leukocyte-stimulating cells (ENLST™ cells)

[0331] As used herein, the term "ENLST™ cell" refers to engineered leukocyte-stimulating cells. According to some embodiments, ENLST™ cells comprise primary tumor cell lines transfected or transduced with a recombinant DNA sequence. According to some embodiments, the recombinant DNA sequence encodes a core of the following essential immunomodulatory peptides: an OX40 ligand (OX40L), a CD27 ligand (CD70), and a CD28 ligand (CD28L) comprising CD80, CD86, or both. According to some embodiments, the recombinant sequence may include one or more additional subsets of immunomodulators designated as R groups (similar to those of the core chemical structure), each subset comprising 3 to 25 immunomodulators.

[0332] According to some embodiments, the method further comprises developing a clone cell bank of ENLST™ cell population(s) to minimize cell heterogeneity. According to some embodiments, ENLST™ is a clone.

[0333] Tumor cell lines

[0334] Tumor-specific antigens

[0335] According to some embodiments, the present disclosure provides a population of ENLST™ cells expressing one or more tumor-specific antigens. According to some embodiments, the tumor-specific antigen may be encoded by a primary open reading frame of a gene product that is differentially expressed by the tumor rather than normal tissue. According to some embodiments, the tumor-specific antigen may be encoded by a mutated gene, an intron sequence, or a translated alternative open reading frame, a pseudogene, or an antisense strand, or may represent the product of a gene translocation event. According to some embodiments, the tumor cells provide a wide range of tumor-specific antigens, many of which are not known in character. According to some embodiments, the tumor antigen is a neoantigen.

[0336] Examples of tumor-specific antigens are, without limitation: (a) non-mutated shared antigens ( for example , melanoma-associated antigen (MAGE), B-melanoma antigen (BAGE), renal tumor antigen (RAGE), and cancer testicular antigen ( for example (b) differentiated antigen ( , NY-ESO), (b) differentiated antigen ( for example (c) prostate-specific membrane antigen [PSMA] and prostate-specific antigen (PSA) in prostate carcinoma, Mart1 / MelanA and tyrosinase present in many melanomas, and carcinogen embryonic antigen (CEA) present in a large percentage of colon cancers, which are antigens present in tissue-restricted and lineage-specific tumor cells; (c) mutated oncogenes and tumor suppressor genes providing novel epitopes for immune recognition ( for example, mutated ras, rearranged bcr / abl, mutated p53); (d) unique idiotype( for example , immunoglobulin antigens in myeloma and B-cell myeloma, T-cell receptors (TCRs) expressed in CTCL), (e) oncolytic virus-derived epitopes ( for example , human papillomavirus-encoded E6 and E7 proteins, Epstein-Barr virus-associated antigens present in primary brain lymphomas); and (f) unmutated oncofetoproteins such as CEA, α-fetoprotein and survivin. According to some embodiments, tumor-specific antigens are selected from antigens listed in publicly available cancer antigen peptide databases (the worldwide web at caped.icp.ucl.ac.be / Peptide / list, the whole of which is incorporated herein by reference). According to some embodiments, tumor-specific antigens include the antigens presented in Table 4 shown below.

[0337] [Table 4]. Tumor-specific antigens.

[0338]

[0339]

[0340]

[0341]

[0342]

[0343] According to some embodiments, the population of tumor cells is derived from cancers selected from the group consisting of melanoma, colorectal carcinoma, leukemia, chronic myeloid leukemia, prostate cancer, head and neck cancer, squamous cell carcinoma, tongue cancer, laryngeal cancer, tonsil cancer, hypopharyngeal cancer, nasopharyngeal cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, hepatocellular carcinoma, glioblastoma and brain cancer.

[0344] According to some embodiments, the tumor cell population is characterized by the expression of one or more of the preferentially expressed antigens of gp100, tyrosinase, Melan-A, tyrosinase-associated protein (TRP-2-INT2), melanoma antigen-1 (MAGE-A1), NY-ESO-1, melanoma (PRAME) CDK4, and multiple myeloma oncogene 1 (MUM-1).

[0345] According to some embodiments, a population of colorectal cancer tumor cells is characterized by the expression of one or more of carcinoembryonic antigen (CEA), MAGE, HPV, human telomerase reverse transcriptase (hTERT), EPCAM, PD-1, PD-L1, p53, and cell surface-associated mucin 1 (MUC1).

[0346] Immunological antigen specificity can arise from one or more amino acid sequences of the antigen, the degree of expression of the corresponding antigen by tumor cells, post-translational modification of the antigen, etc.

[0347] Immunological antigenic specificity for a specific type of cancer cell can also arise from one or more specific fingerprints of multiple tumor antigens, the fact that a specific antigen is expressed by various tumor cells but has a specific use in immunotherapy for a smaller number of tumor types, the fact that a specific set of MHC class I presentable and MHC class II presentable epitopes is present on a specific polypeptide or polypeptide fragment, and by omitting one or more peptides capable of inducing immune tolerance. A skilled technician, for example Relevant nucleic acid and polypeptide sequences can be found on the U.S. government website, ncbi.nlm.nih.

[0348] According to some embodiments, tumor cells are derived from a sample from the subject. According to some embodiments, tumor cells are derived from a tumor cell line or a tumor cell line variant.

[0349] According to some embodiments, the tumor antigen specificity of the described invention may be determined by a parent tumor cell line or a tumor cell line variant selected for modification as an immunomodulator.

[0350] Mother cell line

[0351] According to some embodiments, a tumor cell line or a tumor cell line variant is an open source ( for example DCTD Tumor Repository operated by NIH, Charles River Laboratories Inc., or commercial sources ( for example It may be derived from a cell line established in any one of ATCC, Sigma Alrich, Thermo Fischer Scientific, Genescript, DSM2). According to some embodiments, a new cell line may be newly established from tumor cells derived from a tumor of a cancer patient.

[0352] According to some embodiments, cancer tissue, cancer cells, cells infected with a carcinogen, other pre-tumor cells, and cell lines of human origin may be used as sources. According to some embodiments, the cancer cells may be from established tumor cell lines or tumor cell line variants, e.g., without limitation, established non-small cell lung carcinoma (NSCLC), bladder cancer, melanoma, ovarian cancer, renal cell carcinoma, prostate carcinoma, sarcoma, breast carcinoma, squamous cell carcinoma, head and neck carcinoma, hepatocellular carcinoma, pancreatic carcinoma, or colon carcinoma cell lines.

[0353] According to some embodiments, the established cell line comprises the LNCaP clone FGC (ATCC CRL-1740), which itself originates from metastatic prostate cancer that has migrated to the lymph nodes. According to some embodiments, the established cell line comprises the PC-3 (ATCC CRL-1435) cell line, which itself originates from metastatic prostate cancer that has migrated to the bone. According to some embodiments, the tumor cell line or tumor cell line variant is derived from one or more of the following ATCC cell lines: VCaP (ATCC CRL-2876); MDA PCa 2b (ATCC CRL-2422); or DU 145 (ATCC HTB-81).

[0354] According to some embodiments, the established cell line includes an SK-MEL-2 clone (ATCC HTB-68), which itself originates from a metastasis to the skin of the thigh.

[0355] According to some embodiments, the established cell lines comprise one or more of the breast carcinoma cell lines designated as COO-G, DU4475, ELL-G, HIG-G, MCF / 7, MDA-MB-436, MX-1, SW-613, and VAN-G. According to some embodiments, the established cell lines comprise one or more of the alveolar soft tissue sarcoma cell lines designated as ASPS and ASPS-1. According to some embodiments, the established cell lines comprise one or more lung cell lines designated as LX-1, COS-G, H-MESO-1, H-MESO-1A, NCI-H23, and NCI-H460. According to some embodiments, the established cell lines comprise one or more colon cancer cell lines designated as CX-5, GOB-G, HCC-2998, HCT-15, KLO-G, KM20L2, MRI-H-194, LOVO I, LOVO II, and MRI-H-250. According to some embodiments, the established cell lines comprise one or more melanoma cell lines designated as NIS-G, TRI-G, WIL-G, MRI-H-121B, MRI-H-187, MRI-H-221, and MRI-H-255. According to some embodiments, the established cell lines comprise one or more cervical cancer cell lines designated as MRI-H-177, MRI-H-186, MRI-H-196, and MRI-H-215. According to some embodiments, the established cell lines comprise one or more renal cancer cell lines designated as MRI-H-121 and MRI-H-166. According to some embodiments, the established cell lines comprise one or more endometrial cancer cell lines designated as MRI-H-147 and MRI-H-220. According to some embodiments, the established cell lines comprise one or more ovarian cancer cell lines designated as MRI-H-258, MRI-H-273, MRI-H-1834, and SWA-G. According to some embodiments, the established cell lines include one or more bronchial cell lines designated as HS-1, OGL-G, and DEL-G.According to some embodiments, the established cell line comprises an epidermal cell line designated as DEAC-1. According to some embodiments, the established cell line comprises a glioblastoma cell line designated as SF 295. According to some embodiments, the established cell line comprises a prostate cancer cell line designated as CWR-22. According to some embodiments, the established cell line comprises a Burkitt lymphoma cell line designated as DAU. According to some embodiments, the above-mentioned established cell line described herein is. for example It is commercially available from the American Type Culture Collection (ATCC), the European Collection of Cell Cultures (ECACC), or any depository listed as an International Depository Authority (IDA) under Article 7 of the Treaty of Budapest.

[0356] According to some embodiments, exemplary established cell lines include one or more of the cell lines in the following table:

[0357] [Table 5]. Cell line.

[0358]

[0359]

[0360]

[0361] According to some embodiments, the selection of a parent cell line from which a tumor cell line or tumor cell line variant may be derived may affect the immune specificity of a given engineered leukocyte stimulator cell. For example, the use of a tumor cell line or tumor cell line variant derived from metastatic prostate cancer that has migrated to a patient's bone may result in ENLST™ cells that induce an immune response specific to metastatic prostate cancer in the patient's bone.

[0362] According to some embodiments, a tumor cell line or tumor cell line variant may be derived from a parent cell containing universal cancer-specific antigens. For example, the use of a parent tumor cell line or tumor cell line variant derived from metastatic prostate cancer that has migrated to a patient's bone may result in ENLST™ cells that induce an immune response against all prostate cancer cells.

[0363] According to some embodiments, the tumor cell line or tumor cell line variant is derived from patient-derived cells from various cancers. According to some embodiments, fresh tissue surgically removed from a tumor is enzymatically digested by type IV collagenase, followed by the collection of degraded cells. According to some embodiments, the degraded cells may then be grown in vitro in a growth medium containing 10% fetal bovine serum on an extracellular matrix material, such as collagen or fibronectin, to promote adhesion. According to some embodiments, the adherent cells may then be passaged until the immortal cancer cells outnumber the non-cancer fibroblast cells.

[0364] For example, according to some embodiments, a tumor cell line or a tumor cell line variant may originate from a solid tumor comprising tumor cells comprising cancer stem cells, metastatic cancer comprising metastatic tumor cells comprising cancer stem cells, or non-metastatic cancer. According to some embodiments, the cancer may originate from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testicle, tongue, or uterus. According to some embodiments, the cancer may be of a histological type, for exampleCancer that originates in the tissue lining or covering the skin or internal organs (carcinoma); cancer that originates in the body's bones or soft tissues, including cartilage, fat, muscle, blood vessels, and fibrous tissue (sarcoma); cancer that originates in blood-forming tissues (leukemia); cancer that originates in cells of the immune system (lymphoma); cancer that arises from plasma cells (myeloma) or may be brain / spinal cord cancer.

[0365] Examples of carcinomas include, without limitation, giant cell and spindle cell carcinomas; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; mammary gland carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma of adenomatous polyps; adenocarcinoma, familial polyposis E. coli; solid carcinomas; carcinoid tumors; branched-alveolar adenocarcinoma; papillary adenocarcinoma; chromophore carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; non-encapsulated sclerosing carcinoma; adrenocortical carcinoma; endometrial carcinoma; carcinoma of cutaneous adnexa; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; Includes mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; tension ring cell carcinoma; infiltrative ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, which is mammary gland acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; Sertoli cell carcinoma; embryonic carcinoma; choriocarcinoma.

[0366] Examples of sarcomas include, without limitation, glomerulosarcoma; sarcoma; fibrosarcoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonic rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; carcinosarcoma; synovial sarcoma; angiosarcoma; Kaposi's sarcoma; lymphangiosarcoma; osteosarcoma; paracortical osteosarcoma; chondrosarcoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; odontosarcoma, dechromosomal dentate; melanoblastoma, malignant; dechromosomal fibrosarcoma; myelosarcoma; and mast cell sarcoma.

[0367] Examples of leukemia include, without limitation, leukemia; lymphocytic leukemia; plasma cell leukemia; erythrocytic leukemia; lymphosarcomatous leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocyte leukemia; mast cell leukemia; megakaryocytic leukemia; and blastocyst leukemia.

[0368] Examples of lymphomas and myelomas include, without limitation, malignant lymphoma; Hodgkin's disease; Hodgkin's disease; paragranulomas; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specific non-Hodgkin lymphomas; malignant melanoma; apigmented melanoma; superficial diffuse melanoma; malignant melanoma of giant pigmented nevus; epithelial melanoma; multiple myeloma.

[0369] Examples of brain / spinal cord cancers include, without limitation, pineal tumor, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrinofibrous astrocytoma; astroblastoma; glioblastoma; oligodendrocyte glioma; oligodendrocyte blastoma; primitive neuroectodermal tumor; cerebellar sarcoma; ganglion neuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neuritis, malignant.

[0370] Examples of other cancers include, without limitation, thymoma; ovarian stromal tumor; coma; granulosa cell tumor; androcyte tumor; Reidyg's cell tumor; lipid cell tumor; paraganglioma; extramammary-extraganglioma; pheochromocytoma; blue nevus, malignant; fibrous histiocytoma, malignant; mixed tumor, malignant; Müller mixed tumor; nephroblastoma; hepatoblastoma; mesenchymal tumor, malignant; Brenner tumor, malignant; phyllodes tumor, malignant; mesothelioma, malignant; germ cell tumor; teratoma, malignant; ovarian stromal tumor, malignant; mesonephroma, malignant; hemangioma, malignant; perivascular cell tumor, malignant; chondroblastoma, malignant; granulosa cell tumor, malignant; malignant histiocytosis; and immunoproliferative small bowel disease.

[0371] For any given tumor type, several tumor cell lines or tumor cell line variants may be commercially available. According to some embodiments, pooling of several of these cell lines as a mixture of whole cells or by preparing membrane preparations other than a mixture of whole cells may provide an array of cell surface tumor antigens for that tumor type.

[0372] According to some embodiments, tumor cells or tumor cell lines or tumor cell line variants may be rendered inoperable by radiation.

[0373] Exogenous immunomodulatory molecules

[0374] According to some embodiments, the exogenous immunomodulatory molecule of the disclosed invention is a polypeptide that mediates the stimulation of immune cells when incorporated into a population of ENLST™ cells, either alone or in combination with other exogenous immunomodulatory molecules.

[0375] According to some embodiments, the exogenous immunomodulatory molecule of the disclosed invention is a polypeptide that mediates the stimulation of T-lymphocytes, natural death (NK) cells, dendritic cells (DCs), or B lymphocytes, either alone or in combination with other exogenous immunomodulatory molecules. According to some embodiments, the NK cells are memory-like NK cells. According to some embodiments, the T lymphocytes are cytotoxic T-lymphocytes (CTLs) (CD8+ T cells). According to some embodiments, the T lymphocytes are memory T cells. According to some embodiments, the T lymphocytes are regulatory T cells. According to some embodiments, the T lymphocytes are helper T cells. According to some embodiments, the B lymphocytes are memory B cells. According to some embodiments, it is a feature of the present invention that a population of tumor cells comprising at least three core exogenous immunomodulatory molecules is effective in stimulating one or more types of immune cells, for example, that allogeneic ENLST™ cells comprising the population of tumor cells of the present disclosure are effective in stimulating one or more of T-lymphocytes (e.g., CD8+ T cells), natural death (NK) cells, dendritic cells (DC), or B lymphocytes.

[0376] According to some embodiments, the expression “stimulating immune cells” refers to the activation of immune cells. According to some embodiments, “stimulating immune cells” refers to the expansion of immune cells. According to some embodiments, “stimulating immune cells” refers to an increase in the cytotoxicity of immune cells. According to some embodiments, “stimulating immune cells” refers to one or more combinations of the activation, expansion, and / or increased cytotoxicity of immune cells. According to some embodiments, an ENLST™ cell population of tumor cells comprising at least three core exogenous immunomodulatory molecules is effective in activating and / or expanding immune cells (e.g., T-lymphocytes (e.g., CD8+ T cells), natural death (NK) cells, dendritic cells (DC), or B lymphocytes) in vitro. According to some embodiments, an ENLST™ cell population of tumor cells comprising at least three core exogenous immunomodulatory molecules is effective in activating and / or expanding immune-dead cells (e.g., T-lymphocytes (e.g., CD8+ T cells), natural death (NK) cells, dendritic cells (DC), or B lymphocytes) in vivo. An assay for detecting whether an ENLST™ cell population of tumor cells comprising at least three core exogenous immunostimulatory molecules is effective in stimulating an immune-dead cell population is described herein. According to one aspect, the present disclosure thus provides an ENLST™ cell population comprising a population of tumor cells genetically engineered to express one or more tumor-specific antigens and stably express a plurality of immunomodulatory molecules effective in stimulating one or more T lymphocytes (e.g., CD8+ T cells), natural death (NK) cells, dendritic cells (DC), or B lymphocytes.

[0377] According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises at least three stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes. According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises at least four stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes. According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises at least five stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes. According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises at least six stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes.According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises at least seven stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes. According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises at least eight stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes. According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises at least nine stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes. According to some embodiments, the ENLST™ cell population comprises a population of p tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises at least 10 stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes.According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises at least 11 stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes. According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises at least 12 stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes. According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises at least 13 stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes. According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises at least 14 stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes.According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises at least 15 stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes. According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises at least 16 stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes. According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises at least 17 stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes. According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises at least 18 stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes.According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises at least 19 stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes. According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises at least 20 stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes. According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises at least 21 stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes. According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises at least 22 stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes.According to some embodiments, the ENLST™ cell population comprises a population of t tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises at least 23 stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes. According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises at least 24 stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes. According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises at least 25 stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes. According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises at least 26 stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes.According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises at least 27 stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes. According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises at least 28 stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes. According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises at least 29 stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes. According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises at least 30 stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes.

[0378] According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC) or B lymphocytes, and the population is characterized by the expression of three intrinsically stable expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC) or B lymphocytes, wherein the three intrinsically stable expressed exogenous immunomodulatory molecules are GMCSF, OX40L and 4IBB-L.

[0379] According to some embodiments, exogenous immunomodulatory molecules constituting an additional R subset comprising 3 to 25 comprehensive immunomodulators may be particularly selected from the group for the ability to initiate and / or maintain an anti-tumor immune response, and / or the ability to abolish existing immunosuppression characteristically present in cancer patients, or a combination of all three. According to some embodiments, the combination of immunomodulatory molecules is evaluated and selected based on human mixed lymphocyte tumor cell responses. According to some embodiments, exemplary classes of exogenous immunomodulatory molecules include cytokines, TNF-family members, secreted receptors, chaperones, IgG superfamily members, and chemokine receptors or other immunomodulatory molecules.

[0380] According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises a plurality of stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, wherein the exogenous immunomodulatory molecules comprise one or more cytokine family member proteins and one or more TNF family member proteins; wherein the exogenous immunomodulatory molecules comprise one or more cytokine family member proteins and one or more secreted receptor proteins; wherein the exogenous immunomodulatory molecules comprise one or more cytokine family member proteins and one or more chaperone proteins; wherein the exogenous immunomodulatory molecules comprise one or more cytokine family member proteins and one or more IgG superfamily member proteins; Here, the exogenous immunomodulatory molecule includes one or more cytokine family member proteins and one or more chemokine receptor proteins.

[0381] According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises a plurality of stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, wherein the exogenous immunomodulatory molecules comprise one or more TNF family member proteins and one or more secreted receptor proteins; wherein the exogenous immunomodulatory molecules comprise one or more TNF family member proteins and one or more chaperone proteins; wherein the exogenous immunomodulatory molecules comprise one or more TNF family member proteins and one or more IgG superfamily member proteins; and wherein the exogenous immunomodulatory molecules comprise one or more TNF family member proteins and one or more chemokine receptor proteins.

[0382] According to some embodiments, the ENLST™ cell population comprises a population of t tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC) or B lymphocytes, and the population comprises a plurality of stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC) or B lymphocytes, wherein the exogenous immunomodulatory molecules comprise one or more secreted receptor proteins and one or more chaperone proteins; wherein the exogenous immunomodulatory molecules comprise one or more secreted receptor proteins and one or more IgG superfamily member proteins; and wherein the exogenous immunomodulatory molecules comprise one or more secreted receptor proteins and one or more chemokine receptor proteins.

[0383] According to some embodiments, the ENLST™ cell population constituting the present disclosure comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises a plurality of stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, wherein the exogenous immunomodulatory molecules comprise one or more chaperone proteins and one or more IgG superfamily member proteins; wherein the exogenous immunomodulatory molecules comprise one or more chaperone proteins and one or more chemokine receptor proteins.

[0384] According to some embodiments, the ENLST™ cell population constituting the present disclosure comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, and the population comprises a plurality of stably expressed exogenous immunomodulatory molecules effective in stimulating T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes, wherein the exogenous immunomodulatory molecules comprise one or more IgG superfamily member proteins and one or more chemokine receptor proteins.

[0385] Exemplary immunomodulators are shown in Table 6 below. According to some embodiments, the exogenous immunomodulatory molecule within the R subset is selected from one or more of TNF-family members, secreted receptors, chaperone proteins, IgG superfamily members, and chemokine receptors. According to some embodiments, the TNF-family member is selected from the TNF-family members listed in Table 6. According to some embodiments, the secreted receptor is selected from the secreted receptors listed in Table 6. According to some embodiments, the chaperone protein is selected from the chaperone proteins listed in Table 6. According to some embodiments, the IgG superfamily member is selected from the IgG superfamily members listed in Table 6. According to some embodiments, the chemokine receptor is selected from the chemokine receptors listed in Table 6. According to some embodiments, the exogenous immunomodulatory molecule is derived from a mouse. According to some embodiments, the exogenous immunomodulatory molecule is derived from a human.

[0386] [Table 6]. Exogenous immunomodulatory molecules

[0387]

[0388] According to some embodiments, the exogenous immunomodulatory molecule of Table 6 is in a membrane-bound form (i.e., containing a membrane anchor). According to other embodiments, the exogenous immunomodulatory molecule of Table 6 is in a secreted form. According to some embodiments, the membrane-bound form of the exogenous immunomodulator is one or more selected from the group consisting of 4-1BB ligand, BAFF, April, CD40 ligand, CD80, CD86, Flt3 ligand, GM-CSF, HSP90, ICOS ligand, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL7, LIGHT, OX40 ligand, RANK ligand, and TNF. According to some embodiments, the secreted form of the immunomodulator is one or more selected from the group consisting of Flt3 ligand, GM-CSF, IL10R, IL7, and TGF-beta receptor.

[0389] According to some embodiments, the exogenous immunomodulatory molecule is a molecule having a wild-type amino acid sequence. According to some embodiments, the exogenous immunomodulatory molecule is a molecule having a variant amino acid sequence.

[0390] According to some embodiments, the exogenous immunomodulatory molecule is one or more selected from the group consisting of 4-1BB ligand, APRIL, BAFF, CD27 ligand, CD30L, CD40 ligand, CD80, CD86, FLT-3 ligand, FLT-3 ligand engineered to remove transmembrane regions, GM-CSF, GMCSF engineered with CD8 membrane anchors and IRES-compatible signal sequences, HSP-70, HSP-90, ICOS ligands, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, IL-7 engineered with CD8 membrane anchors, LIGHT, OX-40 ligands, RANK ligands, TGF-β receptors, and TNF.

[0391] According to some embodiments, one or more exogenous immunomodulatory molecules comprise at least three essential immunomodulatory molecules, wherein the three essential immunomodulatory molecules are an OX40 ligand (OX40L), a CD27 ligand (CD70), and a CD28 ligand (CD28L) comprising CD80, CD86, or both. According to some embodiments, an additional immunomodulatory component identified as R may also be present.

[0392] According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens and three stably expressed essential exogenous immunomodulatory molecules, OX40L, CD70, and CD28L, which are effective in stimulating an MNC population. According to some embodiments, an ENLST™ cell population comprising a population of tumor cells expressing one or more tumor-specific antigens and three stably expressed essential exogenous immunomodulatory molecules, OX40L, CD70, and CD80, CD86, or both, and CD28L is effective in stimulating synergistic expansion of CTLs. According to some embodiments, the ENLST™ cell population further comprises one or more subsets of R immunomodulators comprising 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 immunomodulators. According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stably express one R subset comprising CD28L plus 3-25, a comprehensive immunomodulator comprising at least four stably expressed exogenous immunomodulatory molecules OX40L, CD70, and CD80, CD86, or both. According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stably express two R subsets comprising a comprehensive immunomodulator, including at least four stably expressed exogenous immunomodulatory molecules OX40L, CD70, and CD28L plus 3-25, which includes CD80, CD86, or both.According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stably express three R subsets comprising a comprehensive immunomodulator, including at least four stably expressed exogenous immunomodulatory molecules OX40L, CD70, and CD28L plus 3-25, which includes CD80, CD86, or both. According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stably express four R subsets comprising a comprehensive immunomodulator, including at least three essential stably expressed exogenous immunomodulatory molecules OX40L, CD70, and CD28L plus 3-25, which includes CD80, CD86, or both. According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stably express five R subsets including CD28L plus 3-25, a comprehensive immunomodulator, comprising at least three essential stably expressed exogenous immunomodulatory molecules OX40L, CD70, and CD80, CD86, or both. According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stably express six R subsets including CD28L plus 3-25, a comprehensive immunomodulator, comprising at least three essential stably expressed exogenous immunomodulatory molecules OX40L, CD70, and CD80, CD86, or both.According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stably express a 7-R subset comprising a comprehensive immunomodulator, including at least three essential stably expressed exogenous immunomodulatory molecules OX40L, CD70, and CD28L plus 3-25, which includes CD80, CD86, or both. According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stably express an 8-R subset comprising a comprehensive immunomodulator, including at least three essential stably expressed exogenous immunomodulatory molecules OX40L, CD70, and CD28L plus 3-25, which includes CD80, CD86, or both. According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stably express a 9-R subset comprising a comprehensive immunomodulator, including at least three essential stably expressed exogenous immunomodulatory molecules OX40L, CD70, and CD28L plus 3-25, which includes CD80, CD86, or both. According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stably express a 10-R subset comprising a comprehensive immunomodulator, including at least three essential stably expressed exogenous immunomodulatory molecules OX40L, CD70, and CD28L plus 3-25, which includes CD80, CD86, or both.

[0393] According to some embodiments, exogenous immunomodulatory molecule R 1 It is APRIL. According to some embodiments, the exogenous immunomodulatory molecule R2 is BAFF. According to some embodiments, the exogenous immunomodulatory molecule R 3 is a 4-IBB ligand. According to some embodiments, the exogenous immunomodulatory molecule R 4 is a CD30 ligand. According to some embodiments, the exogenous immunomodulatory molecule R 5 is a CD40 ligand. According to some embodiments, the exogenous immunomodulatory molecule R 6 is CD80. According to some embodiments, the exogenous immunomodulatory molecule R 7 is CD86. According to some embodiments, the exogenous immunomodulatory molecule R 8 is a FLT-3 ligand. According to some embodiments, the exogenous immunomodulatory molecule R 9 is HSP-70. According to some embodiments, the exogenous immunomodulatory molecule R 10 is HSP-90. According to some embodiments, exogenous immunomodulatory molecule R 11 is an ICOS ligand. According to some embodiments, the exogenous immunomodulatory molecule R 12 is IL-10R. According to some embodiments, the exogenous immunomodulatory molecule R 13 is IL-12. According to some embodiments, the exogenous immunomodulatory molecule R 14 is IL-15. According to some embodiments, the exogenous immunomodulatory molecule R 15 is IL-18. According to some embodiments, the exogenous immunomodulatory molecule R 16 is IL-2. According to some embodiments, the exogenous immunomodulatory molecule R 17 is IL-21. According to some embodiments, the exogenous immunomodulatory molecule R 18 is IL-23. According to some embodiments, the exogenous immunomodulatory molecule R 19 is IL-7. According to some embodiments, the exogenous immunomodulatory molecule R 20 It is LIGHT. According to some embodiments, the exogenous immunomodulatory molecule R 21is a RANK ligand. According to some embodiments, the exogenous immunomodulatory molecule R 22 is a TGF-β receptor. According to some embodiments, the exogenous immunomodulatory molecule R 23 is TNF. According to some embodiments, the exogenous immunomodulatory molecule R 24 is GM-CSF.

[0394] According to some embodiments, the exogenous immunomodulatory molecule R is selected from the group consisting of APRIL, BAFF, 4-IBB ligand, CD30 ligand, CD80, CD86, FLT-3 ligand, FLT-3 ligand engineered to remove the transmembrane region, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, IL-7 engineered with a CD8 membrane anchor, LIGHT, RANK ligand, TGF-β receptor, and GM-CSF, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, It includes 1 to 30 immunomodulators comprising 23, 24, 25, 26, 27, 28, 29, or 30 exogenous immunomodulatory molecules.

[0395] According to some embodiments, the exogenous immunomodulatory molecule comprises 1 to 30, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, wherein at least three immunomodulatory molecules are an OX40 ligand (OX40L), a CD27 ligand, and a CD28 ligand comprising CD80, CD86, or both, wherein R 1 - R 24Additional immunomodulatory components identified as are selected from the group consisting of APRIL, BAFF, 4-IBB ligand (4-IBBL), CD30L, CD40 ligand, CD80, CD86, FLT-3 ligand, FLT-3 ligand engineered to remove transmembrane regions, GMCSF engineered with CD8 membrane anchor and IRES compatible signal sequence, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, IL-7 engineered with CD8 membrane anchor, LIGHT, RANK ligand, TGF-β receptor, and TNF.

[0396] According to some embodiments, the exogenous immunomodulatory molecule is selected from the group consisting of 4-1BB ligand, APRIL, BAFF, CD27 ligand, CD28 ligand, CD30 ligand, CD40 ligand, CD80, CD86, FLT-3, FLT-3 ligand engineered to remove the transmembrane region, GM-CSF, GMCSF engineered with a CD8 membrane anchor and an IRES-compatible signal sequence, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, IL-7 engineered with a CD8 membrane anchor, LIGHT, OX-40 ligand, RANK ligand, TGF-β receptor, and TNF. in other words It comprises 1 to 20 exogenous immunomodulatory molecules, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. According to some embodiments, the exogenous immunomodulatory molecules are 1 to 20, in other wordsIt comprises exogenous immunomodulatory molecules encompassing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, wherein at least three immunomodulatory molecules are an OX40 ligand (OX40L), a CD27 ligand, and a CD28 ligand, where R 1 - R 24 Additional immunomodulatory components identified as are selected from the group consisting of APRIL, BAFF, 4-IBB ligand, CD30L, CD40 ligand, CD80, CD86, FLT-3 ligand, FLT-3 ligand engineered to remove transmembrane regions, GMCSF engineered with CD8 membrane anchor and IRES compatible signal sequence, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, IL-7 engineered with CD8 membrane anchor, LIGHT, RANK ligand, TGF-β receptor, and TNF.

[0397] According to some embodiments, the exogenous immunomodulatory molecule is selected from the group consisting of 4-1BB ligand, APRIL, BAFF, CD27 ligand, CD28 ligand, CD30 ligand, CD40 ligand, CD80, CD86, FLT-3, FLT-3 ligand engineered to remove the transmembrane region, GM-CSF, GMCSF engineered with a CD8 membrane anchor and an IRES-compatible signal sequence, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, IL-7 engineered with a CD8 membrane anchor, LIGHT, OX-40 ligand, RANK ligand, TGF-β receptor, and TNF. in other words It comprises 1 to 10 exogenous immunomodulatory molecules, including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. According to some embodiments, the exogenous immunomodulatory molecules are 1 to 10, in other wordsIt comprises exogenous immunomodulatory molecules encompassing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, wherein at least three immunomodulatory molecules are OX40 ligand (OX40L), CD27 ligand, and CD28 ligand, where R 1 - R 24 Additional immunomodulatory components identified as are selected from the group consisting of APRIL, BAFF, 4-IBB ligand, CD30 ligand, CD40 ligand, CD80, CD86, FLT-3 ligand, FLT-3 ligand engineered to remove transmembrane regions, GMCSF engineered with CD8 membrane anchor and IRES compatible signal sequence, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, IL-7 engineered with CD8 membrane anchor, LIGHT, RANK ligand, TGF-β receptor, and TNF.

[0398] According to some embodiments, the exogenous immunomodulatory molecules are selected from the group consisting of APRIL, BAFF, 4-IBB ligand, CD30 ligand, CD80, CD86, FLT-3 ligand, FLT-3 ligand engineered to remove the transmembrane region, GM-CSF, GMCSF engineered with a CD8 membrane anchor and IRES-compatible signal sequence, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, IL-7 engineered with a CD8 membrane anchor, LIGHT, RANK ligand, TGF-β receptor, and TNF, 5 to 20, i.e., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or comprises an exogenous immunomodulatory molecule R covering 20. According to some embodiments, the exogenous immunomodulatory molecule comprises 5 to 20, i.e., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, wherein at least three immunomodulatory molecules are an OX40 ligand (OX40L), a CD27 ligand (CD70), and a CD28 ligand (CD28L) comprising CD80, CD86, or both, wherein R 1 - R 24 Additional immunomodulatory components identified as are selected from the group consisting of APRIL, BAFF, 4-IBB ligand, CD30 ligand, CD40 ligand, CD80, CD86, FLT-3 ligand, FLT-3 ligand engineered to remove transmembrane regions, GMCSF engineered with CD8 membrane anchor and IRES compatible signal sequence, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, IL-7 engineered with CD8 membrane anchor, LIGHT, RANK ligand, TGF-β receptor, and TNF.

[0399] According to some embodiments, the exogenous immunomodulatory molecule R comprises 10 to 15, i.e., 10, 11, 12, 13, 14, or 15, selected from the group consisting of APRIL, BAFF, 4-IBB ligand, CD30 ligand, CD80, CD86, FLT-3 ligand, FLT-3 ligand engineered to remove transmembrane regions, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, IL-7 engineered to CD8 membrane anchor, LIGHT, RANK ligand, TGF-β receptor, and TNF. According to some embodiments, the exogenous immunomodulatory molecules comprise 10 to 15, i.e., 10, 11, 12, 13, 14, or 15, wherein at least three immunomodulatory molecules are an OX40 ligand (OX40L), a CD27 ligand (CD70), and a CD28 ligand (CD28L) comprising CD80, CD86, or both, wherein R 1 - R 24 Additional immunomodulatory components identified as are selected from the group consisting of APRIL, BAFF, 4-IBB ligand, CD30 ligand, CD40 ligand, CD80, CD86, FLT-3 ligand, FLT-3 ligand engineered to remove transmembrane regions, GMCSF engineered with CD8 membrane anchor and IRES compatible signal sequence, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, IL-7 engineered with CD8 membrane anchor, LIGHT, RANK ligand, TGF-β receptor, and TNF.

[0400] According to some embodiments, the exogenous immunomodulatory molecules comprise 14 exogenous immunomodulatory molecules selected from the group consisting of APRIL, BAFF, 4-IBB ligand, CD30L, CD80, CD86, FLT-3 ligand, FLT-3 ligand engineered to remove transmembrane regions, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, IL-7 engineered to CD8 membrane anchors, LIGHT, RANK ligand, TGF-β receptor, and TNF. According to some embodiments, the exogenous immunomodulatory molecule comprises 14 exogenous immunomodulatory molecules, wherein at least three immunomodulatory molecules are an OX40 ligand (OX40L), a CD27 ligand (CD70), and a CD28 ligand comprising CD80, CD86, or both, where R 1 - R 24 Additional immunomodulatory components identified as are selected from the group consisting of APRIL, BAFF, 4-IBB ligand, CD30 ligand, CD40 ligand, CD80, CD86, FLT-3 ligand, FLT-3 ligand engineered to remove transmembrane regions, GM-CSF, GMCSF engineered with CD8 membrane anchor and IRES compatible signal sequence, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, IL-7 engineered with CD8 membrane anchor, LIGHT, LIGHT, OX-40 ligand, RANK ligand, TGF-β receptor, and TNF.

[0401] According to some embodiments, each exogenous immunomodulatory molecule 4-1BB ligand, APRIL, BAFF, CD27 ligand, CD28 ligand, CD30L, CD40 ligand, CD80, CD86, FLT-3 ligand, GM-CSF, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, LIGHT, OX-40 ligand, RANK ligand, TGF-β receptor and TNF is a wild-type molecule. According to some embodiments, each exogenous immunomodulatory molecule 4-1BB ligand, APRIL, BAFF, CD27 ligand, CD28 ligand, CD30L, CD40 ligand, CD80, CD86, FLT-3 ligand, GM-CSF, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, LIGHT, OX-40 ligand, RANK ligand, TGF-β receptor, and TNF is a mutant or variant sequence.

[0402] According to some embodiments, exogenous immunomodulatory molecule R 1 It is APRIL. According to some embodiments, the exogenous immunomodulatory molecule R 2 is BAFF. R 3 is a 4-IBB ligand. According to some embodiments, the exogenous immunomodulatory molecule R 4 is CD30L. According to some embodiments, the exogenous immunomodulatory molecule R 5 is a CD40 ligand. According to some embodiments, the exogenous immunomodulatory molecule R 6 is CD80. According to some embodiments, the exogenous immunomodulatory molecule R 7 is CD86. According to some embodiments, the exogenous immunomodulatory molecule R 8 is a FLT-3 ligand. According to some embodiments, the exogenous immunomodulatory molecule R 9is HSP-70. According to some embodiments, the exogenous immunomodulatory molecule R 10 is HSP-90. According to some embodiments, exogenous immunomodulatory molecule R 11 is an ICOS ligand. According to some embodiments, the exogenous immunomodulatory molecule R 12 is IL-10R. According to some embodiments, the exogenous immunomodulatory molecule R 13 is IL-12. According to some embodiments, the exogenous immunomodulatory molecule R 14 is IL-15. According to some embodiments, the exogenous immunomodulatory molecule R 15 is IL-18. According to some embodiments, the exogenous immunomodulatory molecule R 16 is IL-2. According to some embodiments, the exogenous immunomodulatory molecule R 17 is IL-21. According to some embodiments, the exogenous immunomodulatory molecule R 18 is IL-23. According to some embodiments, the exogenous immunomodulatory molecule R 19 is IL-7. According to some embodiments, the exogenous immunomodulatory molecule R 20 It is LIGHT. According to some embodiments, the exogenous immunomodulatory molecule R 21 is a RANK ligand. According to some embodiments, the exogenous immunomodulatory molecule R 22 is a TGF-β receptor. According to some embodiments, the exogenous immunomodulatory molecule R 23 is TNF. According to some embodiments, exogenous immunomodulatory molecule R 24 is a CD86 variant engineered with an IRES-compatible signal sequence. According to some embodiments, the exogenous immunomodulatory molecule R 25 is a FLT3L variant engineered to remove the transmembrane region. According to some embodiments, the exogenous immunomodulatory molecule R 26 is a GM-CSF variant engineered with a CD8 membrane anchor and an IRES-compatible signal sequence. According to some embodiments, the exogenous immunomodulatory molecule R27 is an HSP70 variant engineered with a CD8 membrane anchor. According to some embodiments, the exogenous immunomodulatory molecule R 28 is an HSP-90B1 (GRP94 / 96) variant engineered with a CD8 membrane anchor. According to some embodiments, the exogenous immunomodulatory molecule R 29 is an HSP90 variant engineered with a CD8 membrane anchor. According to some embodiments, the exogenous immunomodulatory molecule R 30 is an ICOSL variant engineered with an IRES-compatible signal sequence. According to some embodiments, the exogenous immunomodulatory molecule R 31 is an IL10R variant engineered to remove the transmembrane region. According to some embodiments, the exogenous immunomodulatory molecule R 32 is an IL-Rα variant engineered to remove the transmembrane region (VSV-GM-CSF tag). According to some embodiments, the exogenous immunomodulatory molecule R 33 is an IL12 variant engineered to be a single chain having a CD8 membrane anchor. According to some embodiments, the exogenous immunomodulatory molecule R 34 is an IL15 variant engineered with a CD8 membrane anchor. According to some embodiments, the exogenous immunomodulatory molecule R 35 is an IL18 variant engineered with a CD8 membrane anchor. According to some embodiments, the exogenous immunomodulatory molecule R 36 is an IL2 variant engineered with a CD8 membrane anchor and an IRES-compatible sequence. According to some embodiments, the exogenous immunomodulatory molecule R 37 is an IL21 variant engineered with a CD8 membrane anchor. According to some embodiments, the exogenous immunomodulatory molecule R 38 is an IL23 variant engineered to be a single chain having a CD8 membrane anchor. According to some embodiments, the exogenous immunomodulatory molecule R 39 is an IL7 variant engineered with a CD8 membrane anchor. According to some embodiments, the exogenous immunomodulatory molecule R 40is a TGFb-R variant engineered to remove the transmembrane region. According to some embodiments, the exogenous immunomodulatory molecule R 41 is a TGFb receptor III variant engineered to remove the transmembrane region. According to some embodiments, the exogenous immunomodulatory molecule R 42 is a mIFNα variant modified to bind to the membrane. According to some embodiments, the exogenous immunomodulatory molecule R 43 is a mIFNαγ variant modified to be membrane-bound. According to some embodiments, the exogenous immunomodulatory molecule R 44 is a cleavage-resistant CD40 ligand (CD40L) variant. Table 7 below shows the R group R 1 -R 44 Presents.

[0403] [Table 7]

[0404]

[0405]

[0406]

[0407] According to some embodiments, at least 12 vectors comprise 14 immunomodulators, wherein 3 essential immunomodulatory molecules are an OX40 ligand (OX40L), a CD27 ligand (CD70), and a CD28 ligand (CD28L) comprising CD80, CD86, or both, and wherein the remaining 11 immunomodulators are R in Table 7 1 - R 44 Selected from. According to some embodiments, at least 11 vectors comprise 14 immunomodulators, wherein 3 essential immunomodulatory molecules are OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L) comprising CD80, CD86, or both, and the remaining 11 immunomodulators are R in Table 7. 1 - R 44Selected from. According to some embodiments, at least 10 vectors contain 14 immunomodulators, wherein 3 essential immunomodulatory molecules are an OX40 ligand (OX40L), a CD27 ligand (CD70), and a CD28 ligand (CD28L) containing CD80, CD86, or both, and wherein the remaining 11 immunomodulators are R in Table 7 1 - R 44 Selected from. According to some embodiments, 14 immunomodulators are selected from Table 6, wherein at least three immunomodulatory molecules are an OX40 ligand (OX40L), a CD27 ligand (CD70), and a CD28 ligand (CD28L) comprising CD80, CD86, or both, and the remaining 11 immunomodulators are R in Table 7. 1 - R 44 Selected from, 14 immunomodulators are in 12 vectors. According to some embodiments, the 14 immunomodulators are selected from Table 6, where three essential immunomodulatory molecules are the OX40 ligand (OX40L), the CD27 ligand (CD70), and the CD28 ligand (CD28L) containing CD80, CD86, or both, and where the remaining 11 immunomodulators are R in Table 7. 1 - R 44 Selected from, wherein 14 immunomodulators are in 11 vectors. According to some embodiments, the 14 immunomodulators are selected from Table 6, wherein three essential immunomodulatory molecules are an OX40 ligand (OX40L), a CD27 ligand (CD70), and a CD28 ligand (CD28L) containing CD80, CD86, or both, and wherein the remaining 11 immunomodulators are R in Table 7 1 - R 44 Selected from, where 14 immunomodulators are in 10 vectors. The vectors may contain additional tags.

[0408] According to some embodiments, the immunomodulator is codon-optimized. "Codon optimization" refers to the modification of codons in a polynucleotide encoding a protein to codons that are used first in a particular organism before others, so that the encoded protein can be expressed more efficiently within it. The genetic code is degenerate because most amino acids are described by multiple codons referred to as "synonyms" or "synonym codons." However, codon usage by a particular organism is not random and is more biased toward specific codon triplets. This bias in codon usage can be much higher in relation to specific genes, genes with common functions or ancestral origins, proteins expressed at high levels versus proteins with low copy numbers, or group protein-coding regions of the organism's genome.

[0409] cytokines

[0410] According to some embodiments, the disclosure comprises an ENLST™ cell population comprising a population of tumor cells genetically engineered to express one or more tumor-specific antigens and one or more R groups of immunomodulatory molecules including a core group of three immunomodulatory molecules plus optionally one or more cytokines, wherein the ENLST™ cell population is effective in stimulating one or more populations of a series of apoptotic cells including T lymphocytes, natural death (NK) cells, dendritic cells (DC), or B lymphocytes. Accordingly, the disclosure comprises cytokines including the full length, fragments, homologs, variants, or mutations of the cytokines. Cytokines include proteins that can affect the biological functions of other cells. Biological functions affected by cytokines may include, but are not limited to, cell growth, cell differentiation, or apoptosis. According to some embodiments, the cytokines of the disclosure may bind to specific receptors on the cell surface so that immune cells ( for example , T lymphocytes( for exampleIt can stimulate CD8+ T cells, natural death (NK) cells, dendritic cells (DC), or B lymphocytes.

[0411] According to some embodiments, the cytokines are granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), Fms-associated tyrosine kinase 3 ligand (FLT3LG), interleukin-1 (IL-1), IL-1a, IL-1b, IL-1ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12p40, IL-12p70, IL-12 / IL-23 P40, IL-13, IL-15, IL-15 / IL15-RA, IL-17, IL-17A, IL-18, IL-21, IL-23, TGF-β, MCP-1, TNF-α and interferon alpha (IFNα), IFNγ, MIP1b, It is selected from Rantes, Tweak, and TREM-1. According to some embodiments, the cytokine is granulocyte-macrophage colony-stimulating factor (GM-CSF). According to some embodiments, the cytokine is Fms-associated tyrosine kinase 3 ligand (FLT3LG).

[0412] According to some embodiments, cytokines are secreted. According to some embodiments, cytokines are membrane-bound.

[0413] Granulocyte-macrophage colony-stimulating factor (GM-CSF)

[0414] Granulocyte-macrophage colony-stimulating factor (GM-CSF; Colony-stimulating factor 2; CSF2) is found in monocytes / macrophages and activated T cells, and acts as a growth factor to stimulate and recruit dendritic cells. GM-CSF is a monomeric glycoprotein secreted by endothelial cells and fibroblasts, as well as cells of the immune system. Human GM-CSF is a 144-amino acid protein containing 17 amino acid signaling peptides that can be cleaved to produce a mature 127-amino acid protein. The biological activity of GM-CSF occurs through binding to heterologous cell surface receptors expressed on monocytes, macrophages, granulocytes, lymphocytes, endothelial cells, and alveolar epithelial cells. The GM-CSF receptor (GM-CSFR) typically has low expression (e.g., 20-200 / cell) but high affinity (Shi Y et al., Granulocyte-macrophage colony-stimulating factor (GM-CSF) and T-cell responses: what we do and don't know, Cell Research (2006) 16: 126-133).

[0415] Melanoma patients treated with soluble GM-CSF as adjuvant therapy demonstrated increased disease-free survival compared to controls. GM-CSF has been used as an immunoadjuvant in various ways without limitation, including the systemic and local application of soluble GM-CSF, GM-CSF fusion proteins, transfection of tumor cells with GM-CSF, and injection of GM-CSF DNA. Recombinant GM-CSF has been used as an adjuvant for various peptide, protein, and viral vaccines and has been found to be an effective adjuvant in patients with melanoma, breast, and ovarian cancer. Fusion proteins containing GM-CSF have also been shown to enhance the immunogenicity of antigens. GM-CSF has been tested for use in gene therapy approaches where allogeneic or autologous GM-CSF-expressing cells are used as vaccines (Kaufman and Wolchok eds., General Principles of Tumor Immunotherapy, Chpt 5, 67-121 (2007)). These treatments have varying degrees of efficacy across several different cancer types.

[0416] According to some embodiments, a tumor cell line or a tumor cell line variant may express the GM-CSF peptide of SEQ ID NO: 13. According to some embodiments, the tumor cell line or a tumor cell line variant may comprise one or more proteins having at least 60% sequence identity with respect to the protein of SEQ ID NO: 13. According to some embodiments, the tumor cell line or a tumor cell line variant may comprise one or more proteins having at least 70% sequence identity with respect to the protein of SEQ ID NO: 13. According to some embodiments, the tumor cell line or a tumor cell line variant may comprise one or more proteins having at least 80% sequence identity with respect to the protein of SEQ ID NO: 13. In some embodiments, the tumor cell line or a tumor cell line variant may comprise one or more proteins having at least 90% sequence identity with respect to the protein of SEQ ID NO: 13. According to some embodiments, a tumor cell line or a tumor cell line variant may comprise one or more proteins having at least 95% sequence identity with respect to the protein of SEQ ID NO: 13. According to some embodiments, a tumor cell line or a tumor cell line variant may comprise one or more proteins having at least 96% sequence identity with respect to the protein of SEQ ID NO: 13. According to some embodiments, a tumor cell line or a tumor cell line variant may comprise one or more proteins having at least 97% sequence identity with respect to the protein of SEQ ID NO: 13. According to some embodiments, a tumor cell line or a tumor cell line variant may comprise one or more proteins having at least 98% sequence identity with respect to the protein of SEQ ID NO: 13. According to some embodiments, a tumor cell line or a tumor cell line variant may comprise one or more proteins having at least 99% sequence identity with respect to the protein of SEQ ID NO: 13.

[0417] According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins comprising a fusion between GM-CSF and HLA-I that enables membrane expression. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 60% sequence identity with respect to the protein of SEQ NO: 42 or SEQ NO: 5. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 70% sequence identity with respect to the protein of SEQ NO: 42 or SEQ NO: 5. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 80% sequence identity with respect to the protein of SEQ NO: 42 or SEQ NO: 5. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 90% sequence identity with respect to the protein of SEQ NO: 42 or SEQ NO: 5. According to some embodiments, a tumor cell line or a tumor cell line variant may comprise one or more proteins having at least 95% sequence identity with respect to the protein of SEQ NO: 42 or SEQ NO: 5. According to some embodiments, a tumor cell line or a tumor cell line variant may comprise one or more proteins having at least 96% sequence identity with respect to the protein of SEQ NO: 42 or SEQ NO: 5. In some embodiments, a tumor cell line or a tumor cell line variant may comprise one or more proteins having at least 97% sequence identity with respect to the protein of SEQ NO: 42 or SEQ NO: 5. According to some embodiments, a tumor cell line or a tumor cell line variant may comprise one or more proteins having at least 98% sequence identity with respect to the protein of SEQ NO: 42 or SEQ NO: 5.According to some embodiments, a tumor cell line or a tumor cell line variant may comprise one or more proteins having at least 99% sequence identity with respect to the protein of SEQ ID NO: 42 or SEQ ID NO: 5.

[0418] Fms-like tyrosine kinase-3 ligand (Flt-3L)

[0419] According to some embodiments, the human Flt3L protein is a membrane-bound hematopoietic tetrahelical bundle cytokine encoded by the FLT3LG gene. Flt3L acts as a growth factor that stimulates the proliferation and differentiation of various blood cell precursors and is important for the production and development of dendritic cells. Mice deficient in Flt3L have low levels of dendritic cells, whereas Flt3L administered to mice or humans results in very high levels of dendritic cells (Shortman et al., Steady-state and inflammatory dendritic-cell development, Nature Reviews Immunology, Vol. 7. 19-30 (2007)).

[0420] According to some embodiments, a subset of R immunomodulators may include a membrane-bound form of Flt-3L. According to some embodiments, ENLST™ cells express the Flt3L peptide of SEQ ID NO: 14. According to some embodiments, ENLST™ cells may be genetically engineered to contain one or more proteins having at least 60% sequence identity with respect to the protein of SEQ ID NO: 14. According to some embodiments, ENLST™ cells may be genetically engineered to contain one or more proteins having at least 70% sequence identity with respect to the protein of SEQ ID NO: 14. According to some embodiments, ENLST™ cells may be genetically engineered to contain one or more proteins having at least 80% sequence identity with respect to the protein of SEQ ID NO: 14. According to some embodiments, ENLST™ cells may be genetically engineered to contain one or more proteins having at least 90% sequence identity with respect to the protein of SEQ ID NO: 14. According to some embodiments, ENLST™ cells may be genetically engineered to contain one or more proteins having at least 95% sequence identity with respect to the protein of SEQ NO: 14. According to some embodiments, ENLST™ cells may be genetically engineered to contain one or more proteins having at least 96% sequence identity with respect to the protein of SEQ NO: 14. According to some embodiments, ENLST™ cells may be genetically engineered to contain one or more proteins having at least 97% sequence identity with respect to the protein of SEQ NO: 14. According to some embodiments, ENLST™ cells may be genetically engineered to contain one or more proteins having at least 98% sequence identity with respect to the protein of SEQ NO: 14. According to some embodiments, ENLST™ cells may be gene...

Claims

Claim 1 A method for preparing a pharmaceutical composition comprising a cell product, wherein the cell product comprises an activated and expanded population of mononuclear cells, wherein the activated and expanded population of mononuclear cells comprises an activated and expanded subpopulation of a series of cytotoxic, apoptotic cells, and wherein the pharmaceutical composition is intended for use in passive immunization of cancer patients who are not currently under the influence of immunosuppressive therapy, and wherein the method comprises the following steps under sterile conditions: (1) providing a population of mononuclear cells (MNCs) isolated from a biological sample; and (2) a step of preparing a population of engineered leukocyte-stimulating cells comprising a population of tumor cells genetically engineered to express one or more tumor-specific antigens and stably express a core group of three immunomodulatory molecules, wherein the core group of the immunomodulatory molecules is an OX40 ligand (OX40L), a CD27 ligand (CD70), and a CD28 ligand (CD28L), wherein the amino acid sequence of the wild-type OX40 ligand comprises SEQ ID NO: 108, the amino acid sequence of the wild-type CD27 ligand comprises SEQ ID NO: 109, and the amino acid sequence of the wild-type CD28 ligand comprises SEQ ID NO: 110, SEQ ID NO: 111, or both; and (3) a step of contacting the population of MNCs of step (1) in a test tube with the engineered leukocyte stimulator cells of step (2) to stimulate the synergistic expansion of a series of cytotoxic apoptotic cells to form a population of activated MNCs, wherein the population of activated MNCs comprises an activated subgroup of a series of cytotoxic apoptotic cells; and (4) a step of expanding the population of activated MNCs comprising an activated subgroup of a series of cytotoxic apoptotic cells in a test tube by culturing the population of activated MNCs to form a cell product containing an activated and expanded population of MNCs comprising an activated and expanded subgroup of a series of cytotoxic apoptotic cells;Here, the above pharmaceutical composition is effective in reducing tumor load.; Claim 2 delete Claim 3 In claim 1, a population of engineered leukocyte-stimulating cells, genetically engineered to express one or more tumor-specific antigens and a core group of three immunomodulatory molecules, is further genetically engineered to express an additional number of immunomodulatory molecules comprising 3 to 25 immunomodulators (“R groups”), wherein the R groups are APRIL, BAFF, 4-IBB ligand, CD30 ligand, CD40 ligand, CD80, CD86, FLT-3 ligand, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, LIGHT, RANK ligand, TGF-β receptor, TNF, a CD86 variant engineered with an IRES-compatible signal sequence, a FLT3L variant engineered to remove a transmembrane region, a CD8 membrane anchor, and an IRES-compatible signal sequence. GMCSF variant, HSP70 variant with CD8 membrane anchor, HSP-90B1 (GRP94 / 96) variant engineered with CD8 membrane anchor, HSP90 variant engineered with CD8 membrane anchor, ICOSL variant engineered with IRES-compatible signal sequence, IL10R variant engineered to remove transmembrane region, IL-Rα variant engineered to remove transmembrane region (VSV-GM-CSF tag), IL12 variant engineered as a single strand with CD8 membrane anchor, IL15 variant engineered with CD8 membrane anchor, IL18 variant engineered with CD8 membrane anchor, IL2 variant engineered with CD8 membrane anchor and IRES-compatible sequence, IL21 variant engineered with CD8 membrane anchor, IL23 variant engineered as a single strand with CD8 membrane anchor, IL7 variant engineered with CD8 membrane anchor, TGFβ-R variant engineered to remove transmembrane region, A method comprising a TGFβ receptor III variant engineered to remove a transmembrane region, a mIFNα variant modified to be membrane-bound, a mIFNαγ variant modified to be membrane-bound, and a CD40L variant resistant to cleavage. Claim 4 A method according to claim 1, wherein the CD28 ligand comprises CD80, CD86, or both. Claim 5 A method according to claim 1, wherein a population of engineered leukocyte-stimulating cells is effective in synergistically inducing 2-log expansion of activated CD8+ cells in peripheral blood mononuclear cells compared to an unmodified control cell line. Claim 6 A method in which, in step (4), the population of activated MNCs is identified and isolated by flow cytometry. Claim 7 The method of claim 1, wherein the population of activated and expanded MNCs comprising a series of cytotoxic, activated and expanded subpopulations of apoptotic cells comprises one or more of an NK cell population, an NKT cell population, a CD8 CTL cell population, a CD4 cell population, and a TCRγδ cell population. Claim 8 In claim 1, the population of the MNC is derived from peripheral blood or umbilical cord blood. Claim 9 In claim 1, the population of the above MNC is autologous tissue for cancer patients, method. Claim 10 In claim 1, the population of the above MNC is homologous to the cancer patient, method. Claim 11 A method according to claim 1, wherein a series of cytotoxic activities of a series of cytotoxic cell subgroups of the activated and extended cytotoxicity are specific to cancer antigens of genetically engineered leukocyte stimulator cells without affecting normal cells. Claim 12 A method according to claim 1, wherein a series of cytotoxic activities of a series of cytotoxic cell subgroups of the activated and extended cytotoxicity are effective in killing cancer cells regardless of cancer type without affecting normal cells. Claim 13 delete Claim 14 delete Claim 15 A cell product comprising a population of activated and expanded mononuclear cells (MNCs), wherein the population of activated and expanded MNCs comprises a series of activated and expanded subpopulations of apoptotic cells that are autotissue-like and cytotoxic to a recipient subject, said cell product being prepared by a process comprising: (a) providing a population of MNCs isolated from a biological sample; (b) preparing a population of engineered leukocyte-stimulator cells comprising a population of tumor cells derived from a primary SK-MEL-2 melanoma tumor cell line that is genetically engineered by transfecting or transducing a recombinant DNA sequence encoding at least three core immunomodulator peptides comprising an OX40 ligand (OX40L), a CD27 ligand (CD70), and a CD28 ligand (CD28L); (c) selecting an engineered leukocyte-stimulator cell clone that stably expresses said at least three core immunomodulator peptides, wherein the amino acid sequence of the wild-type OX40 ligand is Step (d) comprising SEQ ID NO: 108, wherein the amino acid sequence of the wild-type CD27 ligand comprises SEQ ID NO: 109, and the amino acid sequence of the wild-type CD28 ligand comprises SEQ ID NO: 110, SEQ ID NO: 111, or both; Step (d) in vitro contacting the population of MNCs of Step (a) with the engineered leukocyte-stimulating cells of Step (b) in a mixed lymphocyte tumor cell response (MLTR) assay to form a population of activated MNCs comprising an activated subpopulation of a series of cytotoxic apoptotic cells; and Step (e) in vitro expanding the population of activated MNCs comprising an activated subpopulation of a series of cytotoxic apoptotic cells by culturing the population of activated MNCs to form a cell product comprising a population of activated and expanded MNCs, wherein the population of activated and expanded MNCs comprises an activated and expanded subpopulation of a series of cytotoxic apoptotic cells;Here, the cell product is effective in reducing the tumor load.; Claim 16 In claim 15, the population of the activated and expanded MNC comprising a series of cytotoxic, activated and expanded subpopulations of apoptotic cells comprises a CD4 cell population and a CD8 cell cytotoxic T lymphocyte (CTL) population, and one or more of an NK cell population, an NKT cell population, and a TCRγδ cell population, a cell product. Claim 17 In paragraph 15, the above-mentioned cytotoxic series of apoptotic cells is a tumor-destroying cell product. Claim 18 In claim 16, the DNA sequence encoding the wild-type OX40 ligand, the wild-type CD27 ligand, and the wild-type CD28 ligand is a codon-optimized cell product for human expression. Claim 19 In claim 15, the contact at step (d) is effective in synergistically inducing 2-log expansion of CD8+ cells, resulting in a cell product. Claim 20 In paragraph 15, the biological sample is a cell product, which is peripheral blood or umbilical cord blood. Claim 21 A method according to claim 1, further comprising: (5) preparing a unit dose package containing individual doses of the activated and expanded cell product from step (4), freezing the unit dose package, and storing the frozen unit dose package in a cold storage; (6) thawing the frozen unit dose package containing the cell product under controlled conditions; and (7) combining the frozen and thawed cell product with a pharmaceutically acceptable carrier.

Images